Methods and compositions for selectively cleaving DNA containing duplex nucleic acids in a complex nucleic acid mixture, and nuclease compositions for use in practicing the same

ABSTRACT

Methods of selectively cleaving DNA containing duplex nucleic acids in a complex nucleic acid mixture, as well as nuclease containing compositions for use therein, are provided. In the subject methods, a nuclease or composition thereof is employed to provide for selective cleavage of DNA containing duplex nucleic acids in a complex nucleic acid mixture. Also provided are novel duplex-stranded specific nucleases and nucleic acids encoding the same, where the subject nucleases are enzymes that, preferentially cleave deoxyribonucleic acid molecules in perfectly matched nucleic acid duplexes as compared to non-perfectly matched nucleic acid duplexes of the same length and/or single stranded nucleic acids. The subject methods and compositions for practicing the same find use in a variety of different applications, including, but not limited to, nucleic acid analyte detection applications, gene expression profiling applications, detection of nucleic acid variants including single nucleotide polymorphisms applications, preparation of subtracted and normalized nucleic acid libraries, etc. Finally, kits for use in practicing the subject methods are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/US02/38808 filed onDec. 3, 2002; which application claims priority to the filing date ofthe U.S. Provisional Patent Application Ser. No. 60/337,125 filed Dec.4, 2001 and to the filing date of U.S. Provisional Application Ser. No.60/393,699 filed on Jul. 2, 2002; the disclosures of which are hereinincorporated by reference.

INTRODUCTION

1. Field of the Invention

The field of this invention is molecular biology, particularly enzymesand more particularly nucleases.

2. Background of the Invention

Nucleases are enzymes that degrade nucleic acids (e.g., deoxyribonucleicacids, DNA, and ribonucleic acids, RNA) and exist in various biologicalmaterials. These enzymes are involved in DNA and RNA metabolism,including degradation, synthesis and genetic recombination of nucleicacids. Several nucleases are digestive enzymes. Nucleases are generallyclassified into exonucleases and endonucleases according to their modeof action. The former type acts on the terminal of polynucleotide chainof nucleic acid molecule and hydrolyzes the chain progressively toliberate nucleotides, while the latter type cleaves a phosphodiesterbond in nucleic acid molecule distributively to produce DNA or RNAfragments or oligonucleotides.

Among other uses, nucleases find use as reagents in a variety ofprotocols in molecular biology. Because of the ever increasing use ofsuch protocols, there is continued interest in the identification of newnucleases with novel properties. Of particular interest would be theidentification of nucleases and compositions thereof that are capable ofselectively cleaving DNA containing duplex nucleic acids in a complexnucleic acid mixture, as such compositions could be used in methods ofselectively manipulating such duplex nucleic acids and would find use ina variety of different applications in molecular biology and relatedfields. The present invention satisfies this need.

Relevant Literature

Reviews about nucleases and their applications include: Williams R J.Methods Mol Biol 2001; 160:409-429; Meiss G, Gimadutdinow 0, FriedhoffP, Pingoud A M. Methods Mol Biol 2001; 160:37-48; Fors L, Lieder K W,Vavra S H, Kwiatkowski R W. Pharmacogenomics 2000 May; 1(2):219-229;Cappabianca L, Thomassin H, Pictet R, Grange T. Methods Mol Biol 1999;119:427-442; Taylor G R, Deeble J. Genet Anal 1999 February;14(5-6):181-186; Suck D. Biopolymers 1997; 44(4):405-421; Liu Q Y,Ribecco M, Pandey S, Walker P R, Sikorska M. Ann N Y Acad Sci 1999;887:60-76; Liao T H. J Formos Med Assoc 1997 July; 96(7):481-487; SuckD. J Mol Recognit 1994 June; 7(2):65-70; and Liao T H. Mol Cell Biochem1981 Jan. 20; 34(1):15-22.

Articles disclosing nucleases from Arthropoda animals include:Menzorova, et al., Biochemistry (Moscow), vol. 58 (1993) (in Russian)pp. 681 to 691; Menzorova, et al., Biochemistry (Moscow), vol. 59 (1994)pp 321 to 325; Chou & Liao; Biochemica et Biophysica Acta, vol. 1036(1990) pp 95 to 100; Lin et al., Biochemica et Biophysica Acta, vol.1209 (1994) pp 209 to 214; Wang et al., Biochem. J., vol 346 (2000) pp799 to 804.

SUMMARY OF THE INVENTION

Methods of selectively cleaving DNA containing duplex nucleic acids in acomplex nucleic acid mixture, as well as nuclease containingcompositions for use therein, are provided. In the subject methods, anuclease or composition thereof is employed under conditions sufficientto provide for selective cleavage of DNA containing duplex nucleic acidsin a complex nucleic acid mixture. Also provided are novelduplex-specific nucleases (DSN) and nucleic acids encoding the same,where the subject nucleases are enzymes that, under specific cleavageconditions (hereinafter denoted “DSN conditions”), preferentially cleavedeoxyribonucleic acid molecules in perfectly matched nucleic acidduplexes as compared to non-perfectly matched nucleic acid duplexes ofthe same length and/or single stranded nucleic acids. The subjectmethods and compositions for practicing the same find use in a varietyof different applications, including, but not limited to, nucleic acidanalyte detection applications, sequence variant detection applications,gene expression profiling applications, preparation of subtracted andnormalized nucleic acid libraries, etc. Finally, kits for use inpracticing the subject methods are provided.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIG. 1 provides a schematic illustration showing that a novel nuclease(DSN) described in the present application discriminates between shortperfect (cleave) and non-perfect (not cleave) DNA-containing duplexes.

FIG. 2 provides the results of crab DSN action on ss phage M13 DNA andds λ DNA. Lanes 1, 2—negative controls, incubation without nuclease.1—phage M13 DNA alone, 2—mixture containing phage M13 DNA and λ DNA.Lanes 3, 4—digestion of phage M13 and λ DNA mixture by crab nuclease at70° C. for 1.5 min (lane 3) and 5 min (lane 4).

FIG. 3 provides the results of a crab DSN DNAse activity assay performedon ds- and ss-DNA 20-mer DNA substrates labeled by TAMRA at 5′-end andDABCYL at 3′-end. Fluorescence intensity was measured at 570 nm (withexcitation at 550 nm). The relative fluorescence increase in theoligonucleotide substrate, RFI, was defined as RFI=(Fi−Fo/Fmax−Fo)×100%,where Fi is the fluorescence intensity of a substrate after incubationwith nuclease, Fo is the substrate fluorescence in the absence ofenzyme, and Fmax represents the fluorescence of 100% cleaved substrate.For kinetic graph construction, three identical experiments wereperformed and the average values and standard deviations were plotted(first and second sequences shown: SEQ ID NO 55; third sequence shown:SEQ ID NO 56).

FIGS. 4A and B provide results of crab DSN DNAse activity assayperformed on 7,8,9,10, and 20-mer ds-DNA substrates labeled by TAMRA at5′-end and DABCYL at 3′-end. Fluorescence intensity was measured at 570nm (with excitation at 550 nm). The relative fluorescence increase inthe oligonucleotide substrate, RFI, was defined as described in FIG. 3(the first sequence shown in FIG. 4A is labelled 20-mer SEQ ID NO 57;the 7, 8, 9, 10 and 20-mer complements are SEQ ID NOs 58-62,respectively).

FIGS. 5A to C provide results of crab DSN action on onemismatch-containing (A, B) and perfectly matched (C) DNA duplexes formedby 5-carboxyfluorescein (Fl)-5′-gccctatagt-3′-TAMRA probeoligonucleotide and complementary strand. Dotted line—substratefluorescence in the absence of enzyme; firm line—substrate fluorescenceafter incubation with DSN ( labeled probe oligonucleotide in A, B and C,bottom: SEQ ID NO 64; target sequence in A: SEQ ID NO 63; targetsequence in B: SEQ ID NO 65; target sequence in C: SEQ ID NO 66).

FIG. 6 provides a graphic chart of the crab DSN activity fromtemperature. Activity of DNAse was measured using Kunitz assay.

FIG. 7 provides graphic chart of the crab DSN activity from Mg²⁺concentration. To analyze the Mg^(2')-ion dependence of crab DSN, 10 ntlong oligonucleotide labeled with a fluorescent donor(5-carboxyfluorescein) at the 5′end and a quencher (DABCYL) at the3′end, was mixed with non-labeled 18 nt long oligonucleotide thatcontains a region perfectly complemented to 10 nt oligonucleotide toform ds-DNA substrate. Probes were prepared on ice. The DNA endonucleaseactivity was assayed in 20 μl reactions containing 50 mM Tris-HCl,pH8.0; 0.5 mkM of substrate oligonucleotides, 1 Kunitz-units crab DSNand different concentrations of MgCl₂. The reactions were carried out 10min at 35° C. and then stopped by the addition of EDTA solution.Fluorescence was measured on spectrofluorimeter Cary Eclypse (Varian,Australia) in 2 ml dishes.

FIG. 8 provides a schematic diagram of detection of a nucleic acidsequence of interest (gray line) in a complex nucleic acid sample thatalso includes nucleic acids comprising a sequence of interest with onenucleotide change (gray broken line) and nucleic acids without asequence of interest (black line).

FIG. 9 provides a schematic diagram of an DSNP assay with twofluorescence labeled probe oligonucleotides for detection of twosequence variants in nucleic acid samples. Gray circles indicatequenching agent, red and green circles indicate fluorescence donors. Redand green asterisks indicate fluorescence signal that is generated afterfluorescence oligonucleotide cleavage using a nuclease according thesubject invention. Blue circle and square indicate the variablenucleotide position.

FIGS. 10 to 14 provide results obtained from assays demonstrating theability to use a subject nuclease according to the subject invention ina SNP detection application:

FIGS. 10A and B provide results of SNP detection using crab DSN on PCRproducts, FT7normD (A) and TT79cD (B). Blue line and Red line—FT7normDand TT79cD specific oligonucleotide fluorescence before nucleasetreatment, respectively. Black line and Green line—FT7normD and TT79cDspecific oligonucleotide fluorescence after nuclease treatment.

FIG. 11. Detection of 7028 C-T SNP in the COX1 gene with the DSNP assay.PCR fragments of 69, 534 and 952 bp comprising 7028 T or 7028 Cvariants, and pT-Adv plasmids with 69 bp fragment insertions were usedin SNP typing with a T variant-specific signal probe. “DSN+”-reactionwith crab DSN; “DSN-”-control samples, no enzyme. (A) The multi-well PCRplate was photographed with Olympus SZX12 fluorescent stereomicroscopeequipped with a green filter. (B) PCR strips with DSNP results for 69 bpfragments were photographed with Multi Image Light Cabinet (AlphaInnotech Corporation) under UV light.

FIG. 12. Analysis of p53 C309T, prothrombin 20210 G-to-A and MTHFR C677Tpolymorphous sites in homo- and heterozygous DNA by the DSNP assay(scheme 1). (A) Photographs obtained on the fluorescent stereomicroscopeequipped with green (G) and red (R) filters. GR—computer superpositionof images obtained with green and red filters. n/n—homozygous DNAsamples comprising wild-type sequence variant, n/m—heterozygous DNAsamples, m/m—homozygous DNA samples comprising mutant sequence variant.(B) Normalized emission spectra of these samples obtained on aspectrofluorimeter, with excitation at 480 nm (for green fluorescence)and 550 nm (for red fluorescence). Fluorescence values, ΔF, werenormalized as described in Table 1. Green line—homozygous DNA samplescomprising the wild-type sequence variant, red lin—homozygous DNAsamples comprising the mutant sequence, blue line—heterozygous DNAsamples.

FIG. 13. Analysis of Factor V Leiden polymorphism G1691A in homozygousand heterozygous DNA samples by the DSNP assay. Photograph was obtainedon the fluorescent stereomicroscope equipped with green (G) and red (R)filters. GR—computer superposition of images obtained with green and redfilters. n/n—homozygous DNA samples comprising wild-type sequencevariant, n/m—heterozygous DNA samples, m/m—homozygous DNA samplescomprising mutant sequence variant. Non-normalized emission spectra ofthese samples after DSN cleavage reaction were obtained on aspectrofluorimeter, with excitation at 480 nm (for green fluorescence)and 550 nm (for red fluorescence). Green line—homozygous DNA samplescomprising the wild-type sequence variant, red line—homozygous DNAsamples comprising the mutant sequence, blue line—heterozygous DNAsamples.

FIG. 14. Fluorescence intensity data obtained by DSNP assay on DNAsamples containing wild-type and mutant ApoE sequences in differentproportions.

FIG. 15 provides results obtained from assays demonstrating the abilityto use a subject nuclease according to the subject invention in anucleic acid analyte detection in RNA sample. (A) reaction with senseRNA. (B) reaction with antisense RNA (negative control). 1—fluorescenceof the reaction mixture after crab DSN treatment; 2—probe fluorescenceof the reaction mixture without nuclease treatment.

FIG. 16 provides a schematic diagram of a DSN using in theallele-specific amplification methods. Grey circles indicate thequenching agent, green and red circles—fluorescence donors. The greenand red asterisks indicate the fluorescence label generated after probeoligonucleotide cutting by a nuclease according to the subjectinvention. Blue and black squares indicate the universal sequences inthe allele-specific primers and probe oligonucleotides.

FIG. 17 provides a schematic diagram of DSNP assay performed on a solidphase. Shadow rectangles indicate the common part of oligonucleotidesthat cannot be cleaved by a nuclease according to the subject invention.Grey circles indicate the quenching agent, green circles—fluorescencedonors. The green asterisks indicate the fluorescence label generatedafter oligonucleotide cutting by a nuclease according to the subjectinvention.

FIG. 18 provides a schematic diagram of a cDNA equalization procedureaccording to the subject invention. Arrows represent the adapter andcomplementary primer. Dashed lines indicate rare transcripts, blackcolor—abundant transcripts. The scheme shown in FIG. 18 does not showthe ds-cDNA synthesis that might be performed by different ways.

FIG. 19 shows the results of agarose electrophoresis followingethidium-bromide staining of non-normalized amplified cDNA (line 1) andnormalized amplified cDNA (line 2). M-marker, 1 kb ladder (Gibco BRL).

FIG. 20 provides the results of agarose electrophoresis followingethidium-bromide staining of non-normalized amplified liver cDNA (line1), obtained by PCR with SMART II oligonucleotide primer,supernormalized liver cDNA (line 2), 28 cycles; and subtracted livercDNA (line 3), 30 cycles. M-marker, 1 kb ladder (Gibco BRL).

FIG. 21 provides a schematic diagram of an equalizing cDNA subtractionprocedure according to the subject invention. Open boxes represent theadapter and complementary primer.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods of selectively cleaving DNA containing duplex nucleic acids in acomplex nucleic acid mixture, as well as nuclease containingcompositions for use therein, are provided. In the subject methods, anuclease or composition thereof is employed under conditions sufficientto provide for selective cleavage of DNA containing duplex nucleic acidsin a complex nucleic acid mixture. Also provided are novel duplexspecific nucleases and nucleic acids encoding the same, where thesubject nucleases are enzymes that, under “DSN conditions”,preferentially cleave deoxyribonucleic acid molecules containingperfectly matched nucleic acid duplexes as compared to non-perfectlymatched nucleic acid duplexes of the same length and/or single strandednucleic acids. The subject methods and compositions for practicing thesame find use in a variety of different applications, including, but notlimited to, nucleic acid analyte detection applications, sequencevariant detection applications including detection of a singlenucleotide polymorphisms (SNPs), gene expression profiling applications,detection of a specific PCR products, preparation of subtracted andnormalized nucleic acid libraries, etc. Finally, kits for use inpracticing the subject methods are provided.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing the subject components ofthe invention that are described in the publications, which componentsmight be used in connection with the presently described invention.

In further describing the subject invention, the subject methods ofselectively cleaving DNA containing duplex nucleic acids are describedfirst in greater detail. Next, the novel nucleases which find use in thesubject methods and nucleic acid compositions encoding the same, as wellas methods for producing the subject nucleases, antibodies specifictherefore and methods for the generation thereof are fully described.Finally, kits that include the subject nucleases are reviewed.

Methods of Selectively Cleaving DNA Containing Duplex Nucleic Acids

As summarized above, the subject invention provides methods ofselectively cleaving DNA containing duplex nucleic acids in a complexnucleic acid mixture. By “selectively cleaving” is meant that thesubject methods preferentially cut or digest, i.e., cleave,deoxyribonucleic acid molecules present in double-stranded nucleicacids, e.g., DNA-DNA duplexes and DNA-RNA duplexes. In many embodiments,the subject methods are methods of preferentially cleaving perfectlymatched duplex nucleic acids. In other words, the subject methodsprovide for preferential cleavage of perfectly matched duplex nucleicacids, i.e., hybrid structures between perfectly complementary strandswhere no mismatches are present, as compared to non-perfectly matchednucleic acid duplexes of the same length. As such, when practicing thesubject methods on a complex nucleic acid mixture (where the term“complex nucleic acid mixture” refers to a sample that includes two ormore different types of nucleic acids, e.g., single and double strandednucleic acids, RNA and DNA, etc.), perfectly matched DNA containingduplex nucleic acids are cleaved to a much greater extent thannon-perfectly matched nucleic acid duplexes, non-DNA containing nucleicacid duplexes and/or single-stranded nucleic acids. In other words, thesubject methods are able to cleave or cut target DNA containing duplexnucleic acids in a sample at a much greater rate than other nucleicacids that may be present in the sample being treated, where the rate oftarget DNA containing duplex nucleic acid cleavage is typically at leastabout 5 fold, usually at least about 10 fold and more usually at leastabout 50 fold, such as about 100 fold, greater than the rate of cleavageof other nucleic acids that may be present in the sample being treated.

In many embodiments, the DNA containing nucleic acid duplexes that arecleaved by the subject methods are DNA-DNA duplexes that include astretch of perfectly matched complementary nucleic acids of at leastabout 8 bp long, in certain embodiments at least 9 bp long. In otherembodiments, the DNA containing nucleic acid duplexes that are cleavedby the subject methods are DNA-RNA duplexes that include a stretch ofperfectly matched complementary nucleic acids of at least about 13 bplong, in certain embodiments at least 15 bp long.

As the subject methods preferentially cleave DNA in double-strandednucleic acids, the subject methods result in substantially no cleavageactivity with respect to single-stranded nucleic acids. As such, when asample is treated according to the subject methods, the amount of DNAcontaining double-stranded or duplex nucleic acids that is cleaved in acomplex nucleic acid mixture far exceeds the amount of single-strandednucleic acids that is cleaved in the mixture, e.g., by at least about 5fold, usually at least about 10 fold and more usually by at least about50 fold, as measured using the cleavage activity assays brieflysummarized below:

-   1. DSN activity on λ ds DNA and phage M13 ss-DNA is compared by    agarose gel electrophoresis. The reaction is performed in a total    volume of 10 μl comprising 1×DSN buffer (7 mM MgCl₂, 50 mM Tris-HCl,    pH 8.0), 0.6 Kunitz units DSN, 150 ng λ DNA and 50 ng M13 DNA. To    prevent ds structure formation in phage M13 DNA, the reaction    mixture is incubated with active enzyme at 70° C. for 1, 5 or 5 min.    The digestion products are visualized on a 0.9% agarose gel    following ethidium bromide staining.-   2. To compare cleavage rate of the nuclease enzyme on ds and ss DNA,    synthetic oligonucleotide substrates are used. Oligonucleotides    labeled with a fluorescent donor at the 5′ end and a fluorescent    quencher at the 3′ end are used as ss DNA. To generate ds    substrates, labeled oligonucleotides are mixed with equimolar    amounts of complementary non-labeled oligonucleotides. Probes are    prepared on ice. The cleavage reaction is performed in a total    volume of 20 μl comprising 1×DSN buffer, 0.6 Kunitz units DSN, and    0.3 μM oligonucleotide substrate. The reactions are carried out    several times (from 1 sec to 100 h) at 35° C. DNase activity is    evaluated by estimating the change in fluorescence intensity of the    reaction mixture during incubation with DSN. Fluorescence intensity    is measured on a spectrofluorimeter Cary Eclypse (Varian) in 2 ml    dishes. Cleavage curves are plotted to obtain half-time for    substrate cleavage. Half-times of the cleavage for ds DNA and ss DNA    were then compared.

RNase activity is measured essentially as described by Ho et al. (Ho HC., Shiau P. F., Liu F. C., Chung J. G., Chen L. Y. 1998. Purification,characterization and complete amino acid sequence of nuclease C1 fromCunninghamella echinulata var. echinulata. Eur J. Biochem. 256:112-118). The reaction mixture (20 μl) contains 50 mM Tris-HCl (pH 8.0),5 mM MgCl₂, and 0.6 Kunitz units of DSN. After warming the mixture at55° C. for 5 min, freshly prepared RNA (1.2% mass/vol., Baker's yeast,Sigma) is added and the incubation continued for 1 h. Followingincubation, 0.1 volumes of ammonium acetate (7.5 M) and 3 volumes of 96%ethanol are added. The entire solution is mixed and centrifuged. Thesupernatant is diluted five-fold with water and absorbance at 260 nm isdetermined.

As indicated above, the subject methods are methods of selectively orpreferentially cleaving deoxyribonucleic acid (DNA) molecules indouble-stranded nucleic acids. As such, the subject methods are methodsof selectively cleaving DNA in DNA-DNA duplexes, as well as DNA/RNAhybrid duplexes. Therefore, when a sample is treated according to thesubject methods, DNA in DNA containing duplexes are cleaved at a ratethat exceeds the rate of cleavage of any other duplex nucleic acids(e.g., RNA in RNA-RNA and RNA-DNA duplex nucleic acids) that may bepresent in the complex mixture being treated, in many embodiments by atleast about 5 fold, usually at least about 10 fold and more usually byat least about 50 fold, as measured using the cleavage activity assaysdescribed above. The ribo- and deoxyribo-synthetic FRET-labeledoligonucleotides of different length described herein may be used toprepare DNA-DNA, RNA-DNA and RNA-RNA substrates in these assays.

Another feature of the subject methods is that they are methods ofpreferentially cleaving perfectly matched DNA containing duplex nucleicacids (DNA-DNA or DNA-RNA). As such, when a sample is treated accordingto the subject methods, completely matched DNA containing duplexes orcomplexes are cleaved at a rate that is at least about 5 fold, usuallyat least about 10 fold and more usually at least about 50 fold greaterthan the rate at which non-completely matched DNA containing complexes(that include at least one bp mismatch) are cleaved, as measured usingthe cleavage activity assay with fluorescently labeled oligonucleotidesubstrates described above. To prepare nuclease substrates, labeledoligonucleotides may be mixed with non-labeled oligonucleotides to formnon-completely and completely matched duplexes. A schematic diagram ofthe discrimination of the completely matched DNA containing complexesfrom non-completely matched complexes using the subject nucleases isprovided in FIG. 1.

In many embodiments, the discrimination between completely matched andnon-completely matched DNA containing nucleic acid duplexes that arecleaved by the subject methods occurs when these duplexes are DNA-DNAduplexes of at least about 8 bp long, in certain embodiments from 9 to15 bp long and most usually 10 bp long. In other embodiments, thediscrimination between completely matched and non-completely matched DNAcontaining nucleic acid duplexes that are cleaved by the subject methodsoccurs when these duplexes are DNA-RNA duplexes of at least about 12 bplong, in certain embodiments from 13 to 25 bp long and most usually 15bp long.

In practicing the subject methods, a sample to be treated according tothe subject methods is contacted with an appropriate nuclease to providefor the selective cleavage of DNA in DNA containing duplex nucleicacids, as described above.

The subject methods are practiced on nucleic acid samples, i.e., samplesthat include nucleic acids. The samples may be obtained from a varietyof different sources, depending on the particular application beingperformed, where such sources include organisms that comprise nucleicacids, i.e. viruses; prokaryotes, e.g. bacteria, archaea andcyanobacteria; and eukaryotes, e.g. members of the kingdom protista,such as flagellates, amoebas and their relatives, amoeboid parasites,ciliates and the like; members of the kingdom fungi, such as slimemolds, acellular slime molds, cellular slime molds, water molds, truemolds, conjugating fungi, sac fungi, club fungi, imperfect fungi and thelike; plants, such as algae, mosses, liverworts, hornworts, club mosses,horsetails, ferns, gymnosperms and flowering plants, both monocots anddicots; and animals, including sponges, members of the phylum cnidaria,e.g. jelly fish, corals and the like, combjellies, worms, rotifers,roundworms, annelids, molluscs, arthropods, echinoderms, acorn worms,and vertebrates, including reptiles, fishes, birds, snakes, and mammals,e.g. rodents, primates, including humans, and the like. Particularsamples of interest include biological fluids, e.g., blood, plasma,tears, saliva, urine, tissue samples or portions thereof, cells(including cell linear, cell lines, cell cultures etc) or lysatesthereof, etc. The sample may be used directly from its naturallyoccurring source and/or preprocessed in a number of different ways, asis known in the art.

In some embodiments, the sample is treated to provide for linear nucleicacids in the sample, where a number of protocols are known in the art,e.g., mechanical shearing, restriction enzyme digest, etc. In someembodiments, the sample may be from a synthetic source. In manyembodiments, the nucleic acids may be amplified using the methods knownin the art like PCR etc. The PCR methods used in the methods of thepresent invention are carried out using standard methods (see, e.g.,Ausubel et al., Current Protocols in Molecular Biology, John Wiley andSons, New York, 1989; Erlich, PCR Technology, Stockton Press, New York,1989; Innis et al., PCR Protocols: A Guide to Methods and Applications,Academic Press, Harcourt Brace Javanovich, New York, 1990; Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989; Barnes, W. M. (1994)Proc Natl Acad Sci USA, 91, 2216-2220). The primers and oligonucleotidesused in the methods of the present invention are preferably DNA, and canbe synthesized using standard techniques.

In some embodiments, the nucleic acid sample of interest is contactedwith synthetic oligonucleotides to form DNA-containing duplexes. Incertain embodiments, these synthetic oligonucleotides are labeled withlabels known in the art as described in detail below, e.g., thesesynthetic oligonucleotides are labeled with a fluorescent donor andacceptor (or quencher) pair. In some embodiments, these syntheticoligonucleotides are probe oligonucleotides used to detect sequence orsequence variant in nucleic acid(s) in a sample. In this case, eachprobe oligonucleotide can form a perfectly matched duplex with asequence or sequence variant to be detected.

As indicated above, in practicing the subject methods, the sample to betreated is contacted with a sufficient amount of an appropriate nucleaseunder conditions that provide for the selective DNA cleavage in a DNAcontaining duplex nucleic acid, i.e., “DSN conditions.” A variety ofdifferent nucleases may exhibit the specific properties described aboveunder specific cleavage conditions and thus may be employed at least insome of the subject methods.

Representative nucleases of interest include but are not limited to:cation-dependent endonucleases from different sources including DNAase Kfrom Kamchatka crab (Menzorova, et al., Biochemistry (Moscow), vol. 58(1993) (in Russian) pp. 681 to 691; Menzorova, et al., Biochemistry(Moscow), vol. 59 (1994) pp 321 to 325), Ca,Mg dependent endonucleasefrom sea-urchin (Menzorova, N. I., Rasskazov, V. A. Biokhimiia (Rus)1981; vol 46 pp 872 to 880), and members of the family of DNA/RNAnon-specific nucleases like shrimp nuclease (Chou & Liao; Biochemica etBiophysica Acta, vol. 1036 (1990) pp 95 to 100; Lin et al., Biochemicaet Biophysica Acta, vol. 1209 (1994) pp 209 to 214; Wang et al.,Biochem. J., Vol 346 (2000) pp 799 to 804) and the like. Of particularinterest in many embodiments are the duplex-specific nucleases,including the novel nucleases, described below.

In practicing the subject methods, the sample is contacted with asufficient amount of the nuclease being employed under DSN conditionsand is maintained for an amount of time sufficient to provide for thedesired amount of selective cleavage of DNA in duplex nucleic acids. Theamount of nuclease employed will vary depending on the specific nucleasethat is employed. However, in many embodiments, the amount of nucleasethat is employed is one that is from about 5 Kunitz-units/ml to about 80Kunitz-units/ml, usually from about 20 Kunitz-units/ml to about 75Kunitz-units/ml and more usually from about 25 Kunitz-units/ml to about70 Kunitz-units/ml.

In these embodiments, the amount of nuclease employed will varyproportionally as the reaction volume varies. For example, where theactual reaction mixture is double the exemplary reaction mixtureprovided above, the amount of nuclease that is employed is, in certainembodiments, double the exemplary amounts provided above, or is someother proportional amount thereof.

In practicing the subject methods, the temperature of the reactionmixture is typically one that ranges from about 10° C. to about 70° C.,usually from about 15° C. to about 65° C., more usually from about 20°C. to about 60° C. In some embodiments the temperature is a temperaturein which DNA, to be cleaved, forms duplexes. In some embodiments thetemperature is changed during cleavage reaction. For example, at thefirst stage of the cleavage reaction, the temperature is optimal forfragmentation of a sample nucleic acids to short oligonucleotides bysubject nuclease (temperature of fragmentation) and at the second stage,the temperature is optimal for hybridization of sample nucleic acidswith probe oligonucleotides and sufficient for cleavage of all perfectlymatched duplexes generated by target sample nucleic acids and probeoligonucleotides (annealing temperature).

The subject nucleases are employed under conditions (noted as DSNconditions) where these conditions ensure specific cleavage of nucleicacid substrates, as described above. In certain embodiments, “DSNconditions” are conditions in which Mg²⁺ is present. In theseembodiments of DSN conditions, the Mg²⁺ concentration can range fromabout 2 to about 15, where the optimal Mg²⁺ conditions range from about3 to about 12, usually from about 4 to about 10 and more usually fromabout 5 to about 8 mM.

Under certain DSN conditions, the pH typically ranges from about 5 toabout 10, usually from about 7 to about 8.5. In practicing the subjectmethods, the reaction mixture containing the sample of nucleic acids andthe nuclease is typically maintained under DSN conditions for a periodof time ranging from about 1 min to about 48 h, usually from about 10min to about 24 h and more usually from about 20 min to about 2 h.

In practicing the subject methods, the manner in which the variousreagents are contacted with the sample may vary. As such, in certainembodiments, the nuclease may be introduced into the sample after thanthe introduction of any other reagents, e.g., Mg²⁺. In alternativeembodiments, all of the reagents are combined at the same time. In someembodiments, the nuclease may be introduced into the sample before thanthe introduction of some other reagents, e.g., probe oligonucleotides.The manner in which contact or combination is achieved may vary, e.g.,by introducing nuclease into the sample, by introducing an amount ofsample in a nuclease containing medium, etc.

The subject methods are useful in a number of applications in the fieldof genetic analysis. More specifically, the subject methods are usefulin methods for detection and characterization of nucleic acid sequences.In particular, the subject methods find use in applications where onewishes to selectively manipulate, e.g., process, detect, eliminate etc.,DNA containing duplexes in the presence of one or more other types ofnucleic acids, i.e., in a complex nucleic acid mixture. As such, thesubject methods find use in a variety of different applications.

In one type of application, DNA containing perfectly matched duplexnucleic acids is distinguished from other types of nucleic acids thatare present in a complex nucleic acid mixture. In these applications,perfectly matched duplex nucleic acids that include at least onedeoxyribonucleic acid molecule are distinguished from non-perfectlymatched duplex nucleic acids of the same length and from single strandednucleic acids. Specific representative applications of this firstcategory of applications include methods of detecting nucleic acidsequences of interest in a sample and methods of detecting nucleic acidsequence changes in a sample. In another type of application, thesubject methods are employed to selectively remove DNA containing duplexnucleic acids from a sample, e.g., by digestion of such duplex nucleicacids, so as to enrich the sample for nucleic acids that are other thanDNA containing duplex nucleic acids, e.g., to enrich for single strandednucleic acids, e.g., single stranded DNA, single stranded RNA, etc.Specific representative applications of this second category includemethods for construction of subtractive and/or normalized (also denotedas equalized) nucleic acids libraries.

Representative applications of interest include, but are not limited to:methods of detection a nucleic acid analyte(s) of interest in a sample(e.g., methods of identifying bacterial and viral strain nucleic acidanalytes and specie specific nucleic acid analytes in a sample; methodsof expression analysis, methods of the detection of the specific PCRproduct(s), etc.); methods of detection of nucleic acid variantsincluding single nucleotide polymorphisms (SNPs); methods of nucleicacid sequencing; and methods of equalization and subtractivehybridization of nucleic acid samples. Each of these specificapplications employing the subject methods are now described in greaterdetail below.

Methods of Detection of the Sequence(s) and Sequence Variants

The subject methods find use in applications of detection of sequencesor sequence variants in nucleic acid samples. These methods include, butare not limited to: methods of identifying a nucleic acid analyte in asample (e.g., methods of identifying bacterial and viral strain nucleicacid analytes and species specific nucleic acid analytes in a sample;methods of expression analysis, methods of the detection of the specificPCR product(s), etc.); methods of detection of nucleic acid variantsincluding single nucleotide polymorphisms (SNPs); and methods of nucleicacid sequencing.

In the subject methods, the nucleic acid sample to be tested iscontacted with a subject nuclease (e.g., DSN) and with a set of thelabeled short synthetic probe oligonucleotides, that produce perfectlymatched duplexes with different sequences or sequence variants to bedetected. During incubation, perfectly matched duplexes between thenucleic acid sample and probe oligonucleotide are cleaved by the DSN togenerate a sequence-specific signal.

In using the subject methods, the nucleic acid sample to be tested isfirst obtained. As such, the first step in the subject methods is toobtain a nucleic acid sample.

The sample may be obtained from a variety of different sources, as notedabove. The sample may be used directly from its naturally occurringsource and/or preprocessed in a number of different ways, as is known inthe art. Depending on the particular application of interest, nucleicacids in the sample may be RNA, double stranded DNA or single strandedDNA and may be exist in linear as well as circular forms.

In some embodiments, the sample is treated to provide for linear nucleicacids in the sample, where a number of different protocols are known forlinearizing nucleic acids, e.g., mechanical shearing, restriction enzymedigest, etc. In some embodiments, the sample may be from a syntheticsource. In some embodiments, if the sample is RNA sample, the prior stepis first strand cDNA preparation that is performed by any method knownin the art. Depending on the particular interest and the amount of thestarting material, the preparation of amplified DNA might is thenperformed. In other embodiments, nucleic acid sample is RNA that is useddirectly from its naturally occurring source or synthesized (for examplesynthesized from T7 promoter-containing PCR amplified DNA by knownmethods).

In certain embodiments, to decrease the complexity of the nucleic acidsin the sample, amplification of the fraction that is enriched of thenucleic acid sequences to be tested is performed, e.g., by PCR or othermethods known in the art. In certain embodiments, specific PCR primersare used to amplify nucleic acids of interest. In other embodiments,adapter-specific PCR primers are used to amplify nucleic acid sample. Ineach case, PCR primers are constructed to amplify nucleic acid fragmentsthat comprise the site for annealing of probe oligonucleotides. In otherembodiments, the DNA sample is used without amplification.

Thus, in certain embodiments, nucleic acids to be tested are unpurifiedPCR products. In other embodiments, nucleic acids to be tested arenon-amplified DNA and RNA. In some embodiments, nucleic acids to betested are exposed to purification using methods known in the art andethanol precipitation (with following resolution in DSN buffer). Incertain embodiments, the nucleic acids to be tested may vary is size,but typically range in size from about 10 to about 4000 bp (nucleotidebase pairs) long, and in certain embodiments these nucleic acids rangein length from about 50 to about 1000 bp long, such as from about 60 toabout 500 bp long. After the sample is obtained, the nucleic acids ofthe sample are contacted with subject nuclease and with a set of one ormore probe oligonucleotides.The set of probe oligonucleotides may include a separate probeoligonucleotide for each nucleic acid sequence or sequence variant to bedetected. As such, where one is assaying a sample for a single nucleicacid sequence variant, the set of probe oligonucleotides employed mayinclude a single probe oligonucleotide. In other embodiments where oneis assaying for two (or more) different nucleic acid sequence variants,the set may include a different probe oligonucleotide for each of thenucleic acid sequences to be detected. The probe oligonucleotides areDNA in many embodiments. In addition, they may be single-stranded linearmolecules. The probe oligonucleotides may be labeled with a detectablelabel.

Suitable labels include, but are not limited to: fluorescent labels,isotopic labels, enzymatic labels, particulate labels, etc. For example,suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), cyaninedyes, e.g. Cy5, Cy3, BODIPY dyes, e.g. BODIPY 630/650, Alexa542, etc.Suitable isotopic labels include radioactive labels, e.g. ³²P, ³³P, ³⁵S,³H. Other suitable labels include size particles that possess lightscattering, fluorescent properties or contain entrapped multiplefluorophores. The label may be a two stage system, where the target DNAis conjugated to biotin, haptens, etc. having a high affinity bindingpartner, e.g. avidin, specific antibodies, etc. The binding partner isconjugated to a detectable label, e.g. an enzymatic label capable ofconverting a substrate to a chromogenic product, a fluorescent label,and isotopic label, etc.

Of particular interest in many embodiments are oligonucleotide probesthat are fluorescence labeled with two or more different fluorophoresthat are placed in different locations on the probe such that adifferent signal is observed depending on whether the probe is or is notcleave into two or more pieces such that the different positionedfluorophores are separated from each other. Examples of such fluorescentlabels include, but are not limited to: non-FRET fluorescence quenchinglabels, as described in: (1) U.S. Pat. No. 6,150,097(fluorescer-quencher pairs, where fluorescers of interest includeFluorescein, Lucifer Yellow, BODIPY, Eosine, Erythrosine,Tetramethyl-rhodamine, Texas Red and Coumarin and quenchers of interestinclude: DABCYL, DABMI and Malachite Green); the disclosure of which isherein incorporated by reference; (2) self-quenching fluorescent probes,as described in U.S. Pat. No. 6,030,787 (reporter-quencher pairs may beselected from xanthene dyes, including fluoresceins, and rhodamine dyes;naphthylamines, e.g., 1-dimethylaminonaphthyl-5-sulfonate,1-anilino-8-naphthalene sulfonate and 2-p-touidinyl-6-naphthalenesulfonate; 3-phenyl-7-isocyanatocoumarin, acridines, such as9-isothiocyanatoacridine and acridine orange;N-(p-(2-benzoxazolyl)phenyl)-maleimide; benzoxadiazoles, stilbenes,pyrenes, and the like); and; (3) molecular energy transfer probes, asdescribed in U.S. Pat. No. 6,117,635 (where specific fluorophores ofinterest listed in the patent include:4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid; acridine andderivatives, e.g., acridine, acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5, disulfonate(Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide,Brilliant Yellow, coumarin and derivatives, e.g., coumarin,7-amino-4-methylcoumarin (AMC, Coumarin 120),7-amino-4-trifluoromethylcoumarin (Coumarin 151), cyanosine,4′-6-diaminidino-2-phenylindole (DAPI),5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red), 7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin,diethylenetriamine pentaacetate,4-(4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid,4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid,5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL),4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC), eosin andderivatives, e.g., eosin, eosin isothiocyanate, erythrosin andderivatives, e.g., erythrosin B, erythrosin isothiocyanate, ethidium,fluorescein and derivatives, e.g., 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate, QFITC (XRITC), fluorescamine, IR144, IR1446,Malachite Green isothiocyanate, 4-methylumbelliferone, orthocresolphthalein, nitrotyrosine, pararosaniline, Phenol Red,B-phycoerythrin, o-phthaldialdehyde, pyrene and derivatives, e.g.,pyrene, pyrene butyrate, succinimidyl 1-pyrene butyrate, Reactive Red 4(Cibacron.R™ Brilliant Red 3B-A), rhodamine and derivatives, e.g.,6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine,tetramethyl rhodamine isothiocyanate (TRITC), riboflavin, rosolic acid,terbium chelate derivatives); the disclosure of which is hereinincorporated by reference. In these embodiments, the two or moredifferent moieties that make up the detectable label, e.g., thefluorophore moieties, the fluorophore/quencher moieties, etc., arespaced relative to each other to provide for the specific propertiesdesired, as is known in the art, where the spacing required is readilydetermined by those of skill in the art in view of the abovespecifically cited patents.

The amount of sample nucleic acids in the reaction mixture may vary buttypically ranges from 1 mkg/ml to 40 mkg/ml, usually from 2 mkg/ml to 5mkg/ml. The amount of probe oligonucleotides that is contacted with thesample may vary, but typically ranges from about 0.1 mkM to about 1 mkM,usually from about 0.2 mkM to about 0.3 mkM for each target nucleic acidto be detected.

If the nucleic acid sample to be tested is a DNA, the probeoligonucleotides that are contacted with the sample typically range inlength from about 8 to about 60 nucleotides, usually from about 9 toabout 25 nucleotides and more usually from about 10 to about 15nucleotides.

In some embodiments, nucleic acid in the sample is RNA. If the nucleicacid sample to be tested is RNA, the probe oligonucleotides that arecontacted with the sample typically range in length from about 13 toabout 60 nucleotides, usually from about 14 to about 45 nucleotides andmore usually from about 15 to about 25 nucleotides.

The buffer where reaction with the subject nucleases (in certainembodiments, crab DSN) is performed is sufficient to provide forcleavage of DNA-containing perfectly matched duplexes and will varydepending on the specific nuclease that is employed. In certainembodiments, the buffer contains Mg²⁺ in final concentration from 2 toabout 15 mM, more usually from about 6 to about 8 mM. The pH typicallyranges from about 6 to about 10, usually from about 7 to about 8.5.

The amount of the nuclease that is contacted with the nucleic acidsample is sufficient to provide for cleavage of ds-DNA containingsubstrates present or generated in the reaction mixture and will varydepending on the specific nuclease that is employed. In certainembodiments, the amount of enzyme typically ranges 5 Kunitz-units/ml toabout 80 Kunitz-units/ml, usually from about 20 Kunitz-units/ml to about75 Kunitz-units/ml and more usually from about 25 Kunitz-units/ml toabout 70 Kunitz-units/ml.

In some embodiments, the nucleic acid sample is first contacted with thesubject nuclease and the resultant mixture is then maintained attemperature optimal for DSN cleaving for a period of time sufficient toprovide for partial cleavage of ds-DNA and generation of short DNAfragments (on average from about 7 to about 20 nt long). The temperatureof this stage (temperature of fragmentation) is usually from about 50 toabout 65° C., more usually from about 55 to about 60° C. The incubationperiod may vary but typically ranges from about 10 to abpit 30 min.

After this step, the probe oligonucleotide(s) is added, and theresultant mixture is maintained at a temperature sufficient to providethe formation of duplexes between probe oligonucleotides and digestedtarget nucleic acids. The digestion of the perfectly matched duplexesgenerated occurs at the same time. Temperature conditions employed inthis step (annealing temperature) depend, at least in part, on theoligonucleotide length and composition, as is known in the art. Thetemperature typically ranges from about 20 to about 72° C., usually fromabout 25 to about 68° C., and most usually from about 30 to about 40° C.The probe oligonucleotide(s) is contacted with the reaction mixtureeither before, during or after temperature change. In certainembodiments, the nucleic acid sample is contacted with the probeoligonucleotides and the subject nuclease at the same time, andresultant mixture is maintained first at temperature of fragmentationand then at the annealing temperature.

In some embodiments, the step of incubation at a temperature offragmentation is excluded from the protocol. In this case, the nucleicacid sample is contacted with the probe oligonucleotide(s) and thesubject nuclease the same time, and the resultant mixture is maintainedat the annealing temperature for a period of time sufficient forcleavage of any perfectly matched DNA-containing nucleic acids present(or generated) in the reaction mixture by the subject nuclease. Duringincubation, the subject nuclease cleaves the ds DNA containing samplenucleic acids (e.g., PCR products) to generate short DNA fragments thatcan effectively hybridize with probe oligonucleotides. All perfectlymatched duplexes generated by the DNA template and probeoligonucleotides are also cleaved by the subject nuclease. In this casethe temperature conditions typically range from about 20 to about 72°C., usually from about 25 to about 68° C., and most usually from 30 toabout 40° C. The reaction mixture, including the active enzyme, istypically maintained for a period of time ranging from about 30 min toabout 48 hrs, usually from about 2 h to about 24 hrs.

In some embodiments, the cleavage reaction described above is performedin the presence of exonuclease-deficient Klenow fragment (KF(exo-)),which catalyzes strand displacement DNA synthesis by extension of the3′-ends generated in the PCR fragment upon nicking activity of thesubject nuclease. Displaced DNA strands are involved in a reaction withprobe oligonucleotides that results in about 5-20 times increase in thespecific signals generated during probe oligonucleotide cleaving. Inthis case the temperature conditions typically range from about 20 to40° C., usually from about 30 to about 37° C., and most usually from 33to 35° C. The reaction mixture, including the active enzyme, istypically maintained for a period of time ranging from about 30 min toabout 12 hrs, usually from about 1 h to about 2 hrs.

In some embodiments, the nucleic acid sample being treated or assayed isprocessed, e.g., heated, to destroy the secondary structures that arepresent among the nucleic acids of the sample. In this case, the sampleis contacted with the set of probe oligonucleotides and subjected todissociation and annealing conditions. The order in which thedissociation and contacting occurs may be varied, so long as the probeoligonucleotides are combined with the dissociated nucleic acids of thesample prior to the annealing step. As such, the probe nucleic acids maybe contacted with the sample prior to dissociation in certainembodiments. In other embodiments, the probe nucleic acids may becontacted with the sample after dissociation but before annealing. Themanner in which the probe oligonucleotides are contacted with the samplemay vary, where representative protocols including pipette introduction,etc. To denature or dissociate the secondary structures present innucleic acids in the sample, including the duplex target nucleic acidspresent therein, the sample is heated to an elevated temperature and theheated sample is maintained at the elevated temperature for a period oftime sufficient for any double-stranded or hybridized nucleic acidspresent in the reaction mixture to dissociate. For denaturation, thetemperature of the reaction mixture will usually be raised to, andmaintained at, a temperature ranging from about 85 to 100° C., usuallyfrom about 90 to 98° C. and more usually from about 93 to 96° C. for aperiod of time ranging from about 1 to 200 sec or more, often from about3 to 120 sec, usually from about 5 to 60 sec.

Next, the dissociated nucleic acids in the sample are allowed toreanneal in the presence of the probe oligonucleotides, which have beencontacted with the sample either before, during or after dissociation.The resultant reaction mixture is then subjected to conditionssufficient for probe annealing to template complementary nucleic acidpresent in the mixture. The temperature to which the reaction mixture islowered to achieve these conditions will usually be chosen to provideoptimal efficiency and specificity, and will generally range from about20 to about 72, usually from about 25 to 68° C., where the specifictemperature conditions employed depend, at least in part, on theoligonucleotide length, as is known in the art. Annealing conditions maybe maintained for a period of time ranging from about 15 sec toovernight or longer.

Following this annealing step (or during the annealing step—in certainembodiments, the steps of annealing and digestion are united), thereaction mixture is contacted with the subject nuclease under DSNconditions (as described above) for a period of time sufficient for thedouble-stranded DNA containing substrates present in the sample, whichsubstrates include perfectly matched duplexes of target nucleic acidsand oligonucleotide probes, to be cleaved by the enzyme.

At the end of the incubation period, the activity of the nuclease enzymeis quenched, e.g., by addition of nuclease inhibitors, in someembodiments, by addition of a metal ion chelator, such as EDTA etc.,where the amount of inhibitor added to the mixture is sufficient toeffectively quench/inhibit all of the enzyme activity in the mixture. Insome embodiments, the activity of the enzyme is quenched by heating at95-97° C. for 7-10 min. In some embodiments, the activity of the enzymeis not quenched. In this case, the reaction mixture is typically notkept more than two hours before signal detection.

Following the above cleavage step, the presence of cleavedoligonucleotide probes is then detected and related to the corresponding(i.e., the probe's complementary) nucleic acid sequence variant presentin the sample. The manner in which the cleaved oligonucleotide probesare detected necessarily depends on the nature of the oligonucleotideprobe label. For example, where the detectable label is an isotopiclabel positioned at one end of the probe, one can assay for detectablylabeled fragments that are shorter than the full-length probe size and,in this manner, detect the presence of cleaved probes.

In certain embodiments, fluorescence labeled probes that provide for adistinct signal upon cleavage are employed, where examples of suchprobes include the FRET and fluorescence quencher probes describedabove. In these embodiments, the mixture is assayed for the presence offluorescence signal that occurs only upon probe cleavage, and detectionof this unique signal is employed to detect the presence of cleavedprobes.

Since the above conditions result in cleavage of substantially onlyperfectly matched or complementary nucleic acid molecules, cleavage ofpreferably those probes for which a perfectly complementary nucleic acidsequence or sequence variant is present in the sample occurs. As such,the detection of probe cleavage products in the sample, as describedabove, provides for a highly accurate and specific determination ofwhether or not the corresponding nucleic acid sequence or sequencevariant is present in the sample.

As mentioned above, the above general methods can be used to detect thepresence of a single nucleic acid sequence variant of interest in asample or a plurality of different nucleic acid sequences in a sample.When a plurality of different nucleic acid sequence variants are to bedetected, the only limitation is that a labeling system, i.e., signalproducing system of one or more entities, should be employed thatprovides for ready differentiation of different oligonucleotide probesto different target nucleic acid sequence variants. Alternatively, inthe case of the array of the subject invention (described below), theoligonucleotides may contain a same label, because the signals fordifferent sequence variants would be different by their positions.

The oligonucleotide probes that are employed may be in solution orimmobilized on a solid support, e.g., presented as an array ofoligonucleotide probes. The solid supports useful in the methods of theinvention include, but are not limited to, agarose, acrylamide, andpolystyrene beads; polystyrene microtiter plates (for use in, e.g.,ELISA); and nylon and nitrocellulose membranes (for use in, e.g., dot orslot blot assays). Some methods of the invention employ solid supportscontaining arrays of oligonucleotide probes. In these cases, solidsupports made of materials such as glass (e.g., glass plates), siliconor silicon-glass (e.g., microchips), or gold (e.g., gold plates) can beused. Methods for attaching nucleic acid probes to precise regions onsuch solid surfaces, e.g., photolithographic methods, are well known inthe art, and can be used to make solid supports for use in theinvention. (For example, see, Schena et al., Science 270:467-470, 1995;Kozal et al., Nature Medicine 2(7):753-759, 1996; Cheng et al., NucleicAcids Research 24(2):380-385, 1996; Lipshutz et al., BioTechniques19(3):442-447, 1995; Pease et al., Proc. Natl. Acad. Sci. USA91:5022-5026, 1994; Fodor et al., Nature 364:555-556, 1993; Pirrung etal., U.S. Pat. No. 5,143,854; and Fodor et al., WO 92/10092.)

In embodiments where the oligonucleotide probe is immobilized on solidsupport, e.g., through covalent or non-covalent interactions, thepresence of cleaved probe on the support is detected and related to thenucleic acid analyte to which that probe hybridizes, e.g., understringent hybridization conditions, such as the annealing conditionsdescribed above and in the experimental sections, below. For example,where the probe is labeled with a fluorescent label that provides for aunique signal when the probe is cleaved, the signal on the substrate isdetected and related to the presence of the nucleic acid analyte in thesample.

The above methods can be employed to either qualitatively orquantitatively detect the presence of the nucleic acid sequence(s) inthe sample. For example, the detection of cleaved oligonucleotide probesprovides a qualitative determination of the nucleic acid sequences inthe sample. To obtain a quantitative determination of the nucleic acidsequences in the sample, one can include a control in the assay, e.g., aknown about of dsDNA labeled substrate, which provides a reference valueto which the detected signal can be compared and thus extrapolated toprovide a quantitative value.

The above methods find use in a number of different specificapplications. Representative specific applications of interest includethe detection of nucleic acid analytes (e.g. in diagnostics andbiological applications where the presence of one or more nucleic acidanalytes is indicative of the presence of a certain condition, organism,etc., in gene expression profiling where multiple nucleic acid analytesexpressed in a cell are detected and compared to the same set of nucleicacids in a reference cell), detection of nucleic acid sequence variants(e.g. analysis of known point mutations, or single oligonucleotidepolymorphisms in a DNA sample(s), allele discrimination, nucleic acidsequencing, etc.) and the like.

Examples of each of these specific applications are described in greaterdetail in the Experimental Section, infra.

Production of Normalized and Subtracted Libraries and Probes forDifferential Screening

The present invention provides several methods for obtaining subtractiveand/or equalized DNA libraries and for preparation probes fordifferential screening. The methods are based on selectively cleavage ofDNA in DNA containing nucleic acid duplexes to retain the singlestranded DNA of interest. As such, the subject methods can be used forthe elimination of the fractions of redundant and/or common molecules ofDNA during normalization and\or subtractive hybridization.

In using the subject enzymes for normalization and\or subtractivehybridization, the sample from which equalized and\or subtractedlibraries are to be produced is first obtained. The sample may beobtained from a variety of different sources, depending on theparticular application being performed, where such sources as describedin the previous section.

Depending on the particular interest, nucleic acids in the sample may bea RNA, double stranded DNA (for ex. genomic DNA or cDNA), or singlestranded DNA.

If the tester nucleic acids (nucleic acids to be subtracted and/ornormalized) is RNA, the prior step is first strand DNA preparation thatis performed by any method known in the art. Depending on the particularinterest and the amount of the starting material, the preparation ofamplified DNA might is then performed. The DNA samples can also beexposed to mechanical shearing, restriction enzyme digest, etc. However,the methods are applicable for lengthy DNA, e.g. full-length cDNA. Insome embodiments, the sample may be from a synthetic source.

While the DNA preparation can be performed by different standardmethods, the resultant DNA molecules must comprise the terminalsequences of the known structure (adapters) that are used following PCR.Adapters are included in DNA molecules by the method known in the art,for example, during DNA preparation or are ligated to the prepared DNA.

Driver nucleic acids (nucleic acids that can hybridize with the fractionthat must be eliminated from tester nucleic acids) may be a RNA, doublestranded DNA (for ex. genomic DNA or cDNA), or single stranded DNA. Ifthe driver nucleic acids are DNA, they must not contain the adaptersequences. In some embodiments these nucleic acids are DNA fragments oroligonucleotides that can hybridize with the fraction that must beeliminated from tester nucleic acids.

In certain embodiments nucleic acid samples are purified usingappropriate protocols and methods known in the art (e.g. using QIAGENPurification systems) and precipitated by ethanol with followingresolution in the hybridization buffer. After the sample is obtained,the resultant DNA is denatured in the presence (subtractivehybridization or supernormalization)/or absence (normalization) of thedriver nucleic acids obtained from the same (supernormalization) orother (driver for subtractive hybridization, probes for differentialscreening, etc.) sources. Denatured nucleic acids are allowed to anneal(hybridization step). Annealing (sometimes called hybridization) refersto the process by which complementary single-stranded nucleic acids forma double-stranded structure, or duplex, mediated by hydrogen-bondingbetween complementary bases in the two strands. Annealing conditions arethose values of, for example, temperature, ionic strength, pH andsolvent which will allow annealing to occur. Many different combinationsof the above-mentioned variables will be conducive to annealing.

During annealing most of the abundant DNA molecules will formdouble-stranded (ds) molecules, and the single-stranded (ss) fractionwill be equalized to a considerable extent [Galau G. A., Klein W H.,Britten R. J., Davidson E. H. II Arch. Biochem. Biophys. 1977. V. 179.P. 584-599]. In the presence of driver nucleic acids, annealing leads toduplex formation between the driver and tester nucleic acid strands if aparticular sequence is common to both nucleic acid populations.Non-hybridized single-stranded DNA is enriched in sequences present inthe experimental cell or tissue which is related to the particularchange or event being studied.

After the hybridization step, the DNA containing duplex nucleic acidsare digested by a suitable nuclease under DSN conditions (as describedabove) for a period of time sufficient for the DNA containingdouble-stranded substrates present in the sample to be cleaved by theenzyme. The amount of enzyme that is contacted with the reaction mixtureis sufficient to provide for cleavage of DNA containing duplex nucleicacids present therein, where the amount typically ranges from about 5min to about 48 h, usually from about 10 min to about 12 h and moreusually from about 20 min to about 1 h. The temperature of the reactionmixture during this incubation period typically ranges from about 55 toabout 72° C., usually from about 60 to about 65° C. At the end of theincubation period, the activity of the enzyme is quenched, e.g., byheating of the reaction mixture (for example at 97° C. for 7-10 min) orby addition of enzyme inhibitors (for example by addition of a metal ionchelator, such as EDTA etc.), where the amount of inhibitor added to themixture is sufficient to effectively quench/inhibit all of the enzymeactivity in the mixture.

ss-DNA fraction enriched in molecules of interest may be then amplifiedby PCR with adapter-specific primer.

Duplex Specific Nucleases

As summarized above, the subject invention also provides novel nucleasesfor use in practicing the abov-described methods. The subject nucleasespreferentially cleave deoxyribonucleic acids molecules indouble-stranded form. The subject enzymes are endonucleases, such thatthey cut, i.e. cleave; break, etc., deoxyribonucleic acid molecules at apoint other than the end of the molecule, if present, and do not requirethe molecule to have ends, e.g., the molecule may be a closed circularmolecule. The subject enzymes cleave deoxyribonucleic acid substratemolecules in a manner that produces 5′ phosphooligonulceotides, i.e.,they cleave between the phosphate and the 3′ hydroxyl to yield 5′phosphomonoester products.

A feature of the subject nucleases is that, under certain conditions(hereinafter referred to as “DSN conditions”), the subject enzymespreferentially cleave deoxyribonucleic acid molecules in double-strandednucleic acids (duplex-specific nucleases, DSN). Furthermore, under DSNconditions, the subject enzymes cleave deoxyribonucleic acid moleculesin perfectly matched short nucleic acid duplexes with substantiallygreater activity than non-perfectly matched nucleic acid duplexes of thesame length.

As the subject enzymes preferentially cleave double-stranded nucleicacids under DSN conditions, they exhibit substantially no cleavageactivity with respect to single-stranded nucleic acids. As such, thedouble-stranded nucleic acid cleavage activity of the subject enzymesfar exceeds their single-stranded nucleic acid cleavage activity underDSN conditions, where the ds-nucleic acid cleavage activity exceedsss-nucleic acid cleavage activity by at least about 5 fold, usually atleast about 10 fold and more usually by at least about 50 fold, asmeasured using the cleavage activity described in above. The ds-nucleicacids that are cleaved by the subject enzymes are nucleic acids thatcontain at least one deoxyribonucleic acid (DNA) molecule. As such, thesubject enzymes cleave DNA duplexes, as well as DNA in DNA/RNA hybridduplexes. However, the subject enzymes exhibit substantially no cleavageactivity with respect to RNA/RNA duplexes, such that the cleavageactivity of DNA containing duplexes exceeds the cleavage activity of anyother duplex nucleic acids by at least about 10 fold, usually at leastabout 50 fold and more usually by at least about 100 fold, as measuredusing the cleavage activity described above.

The subject enzymes preferentially cleave DNA in perfectly matched DNAcontaining short nucleic acid duplexes (DNA-DNA or DNA-RNA) withessentially greater activity than non-perfectly matched DNA containingnucleic acid duplexes of the same length. As such, under DSN conditions,the subject enzymes cleave completely matched DNA containing complexesat a rate that is at least about 5 fold, usually at least about 10 foldand more usually at least about 50 fold greater than the rate at whichnon-completely matched DNA containing complexes (that include as few asone bp mismatch) are cleaved, as measured using the cleavage activitydescribed above.

The minimal duplex length of the nucleic acid substrates of the subjectnucleases is, in many embodiments, at least 8 bp for DNA-DNA duplexesand 13 bp for DNA-RNA duplexes.

In certain embodiments, the subject nucleases are divalent cationdependent nucleases, such that in the absence of divalent cations, thesubject enzymes are inactive, i.e., they do not cleave nucleic acids. Inmany embodiments, the subject nucleases are divalent metal cationdependent, such that in the absence of divalent metal cations, they donot cleave nucleic acids. As the subject enzymes are divalent cationdependent, their activity in the presence of divalent cations, specificdivalent metal cations, can be inhibited or quenched by metal ionchelators, e.g., EDTA, etc. In addition, in some embodiments, thesubject nucleases are thermostable. By thermostable is meant that thesubject nucleases retain their activity over a wide range of elevatedtemperatures. As such, the subject nucleases are active at temperaturesfrom about 15° C. to about 70° C., and show optimal activity attemperatures ranging from about 25° C. to about 65° C., usually fromabout 30° C. to about 65° C. and more usually from about 50° C. to about60° C.

In some embodiments, the subject nucleases exhibit activity inconditions ranging in pH from about 6 to more than 10, where optimalactivity is found at a pH ranging from about 6 to about 10.

In certain embodiments, the subject nucleases exhibit specific featuresunder certain conditions (DSN conditions). In certain embodiments, “DSNconditions” are conditions in which Mg²⁺ is present. In DSN conditions,the Mg²⁺ concentration can range from about 2 to about 15, where theoptimal Mg²⁺ conditions range from about 3 to about 12, usually fromabout 4 to about 10 and more usually from about 6 to about 8 mM. UnderDSN conditions, the pH typically ranges from about 6 to about 10,usually from about 7 to about 8.5.

In certain embodiments the subject nucleases are from, or have an aminoacid sequence that is substantially the same as or identical to, anuclease having the above properties and found in, a Metazoan animal,particularly an Arthropodoan animal, including the specific animalsprovided in the experimental section below. In certain embodiments, thenucleases are from, or have an amino acid sequence that is substantiallythe same as or identical to nuclease having the above properties andfound in, a crustacean, and more specifically Paralithodes camtschatica(also known as Kamchatka crab; red king crab), or the other specificanimals listed in the experimental section below.

In certain embodiments, the subject enzymes have an amino acid sequencethat is substantially the same as or identical to Kamchatka crabnuclease having the following amino acid sequence:

MANMESKQGIMVLGFLIVLLFVSVNGQDCVWDKDTDFPEDPPLIFDSNLELIRPVLENGK (SEQ ID NO:01) RIVSVPSGSSLTLACSGSELINLGMEAVEAKCAGGVMLAIEGTEWEIWSLGCSNHVKETIRRNLGTCGEADQGDRHSIGFEYYGGSIYYELISVCFGPVSETTLRTEHVLHGANIAAKDIETSRPSFKTSTGFFSVSMSTVYSQASQLQLMTDILGDSDLANNIIDPSQQLYFAKGHMSPDADFVTVAEQDATYYFINALPQWQAFNNGNWKYLEYATRDLAESHGSDLRVYSGGWSLLQLDDINGNPVDILLGLSEGKEVVPVPSLTWKVVYEESSSKAAAIVGINNPHITTAPSPLCSDLCSSLTWIDFNLDDLAHGYTYCCAVDDLRQAIPYIPDLGNVGLLTN

In certain embodiments, the subject enzymes have an amino acid sequencethat is substantially the same as or identical to the specific nucleaseshaving the amino acid sequences provided in the experimental sectionbelow.

By “substantially the same as” is meant a protein having an amino acidsequence that has at least about 30%, sometimes at least about 40%,sometimes at least about 50%, sometimes at least about 60%, sometimes atleast about 75%, and in certain embodiments at least about 80%, at leastabout 90% and in certain embodiments at least about 95%, 96%, 97%, 98%or 99% sequence identity with the sequence of SED ID NO:01, as measuredby the BLAST compare two sequences program available on the NCBI websiteusing default settings using the full length sequence.

In addition to the specific nuclease proteins described above, homologsor proteins (or fragments thereof from other species, i.e., other animalspecies, are also provided, where such homologs or proteins may be froma variety of different types of species. By homolog is meant a proteinhaving at least about 35%, usually at least about 40% and more usuallyat least about 60% amino acid sequence identity to the specific proteinsprovides above, where sequence identity is determined using thealgorithm described supra.

The subject nucleases are present in a non-naturally occurringenvironment, e.g. are separated from their naturally occurringenvironment. In certain embodiments, the enzymes are present in acomposition that is enriched for subject enzymes as compared to theirnaturally occurring environment. As such, purified nucleases areprovided, where by purified is meant that nucleases are present in acomposition that is substantially free of non-nuclease proteins, whereby substantially free is meant that less than 90%, usually less than 60%and more usually less than 50% (by dry weight) of the composition ismade up of non-nuclease proteins.

In certain embodiments of interest, the subject proteins are present ina composition that is substantially free of the constituents that arepresent in their naturally occurring environment. For example, anuclease comprising composition according to the subject invention inthis embodiment is substantially, if not completely, free of those otherbiological constituents, such as proteins, carbohydrates, lipids, etc.,with which it is present in its natural environment. As such, proteincompositions of these embodiments will necessarily differ from thosethat are prepared by purifying the protein from a naturally occurringsource, where at least trace amounts of the constituents or othercomponents of the protein's naturally occurring source will still bepresent in the composition prepared from the naturally occurring source.

The subject proteins may also be present as isolates, by which is meantthat the proteins are substantially free of both non-nuclease proteinsand other naturally occurring biologic molecules, such asoligosaccharides, polynucleotides and fragments thereof, and the like,where substantially free in this instance means that less than 70%,usually less than 60% and more usually less than 50% (by dry weight) ofthe composition containing the isolated protein is a non-deoxyribosenaturally occurring biological molecule. In certain embodiments, thesubject proteins are present in substantially pure form, where bysubstantially pure form is meant at least 95%, usually at least 97% andmore usually at least 99% pure.

In yet other embodiments, the subject nuclease may be present in apreparation that contains much less of the nuclease, e.g., less thanabout 50%, usually less than about 25% and often less than about 10 oreven 5%, where in these embodiments, the preparation may have beentreated, e.g., warmed.

In addition to the naturally occurring proteins described above,polypeptides that vary from the naturally occurring proteins are alsoprovided. Such polypeptides are proteins having an amino acid sequenceencoded by an open reading frame (ORF) of a protein according to thesubject invention, described above, including the full length proteinand fragments thereof, particularly biologically active fragments and/orfragments corresponding to functional domains, and including fusions ofthe subject polypeptides to other proteins or parts thereof. Fragmentsof interest will typically be at least about 10 aa in length, usually atleast about 50 aa in length, and may be as long as 250 aa in length orlonger, but will usually not exceed about the length of the full lengthprotein.

Nucleic Acid Compositions

Also provided are nucleic acid compositions that encode the subjectnucleases, fragments thereof, etc., as described above. Specifically,nucleic acid compositions encoding the above described enzymes/proteins,as well as fragments or homologs thereof, are provided. By “nucleic acidcomposition” is meant a composition comprising a sequence of nucleotidebases that encodes a nuclease polypeptide of the subject invention, asdescribed above, i.e., a region of genomic DNA capable of beingtranscribed into mRNA that encodes a subject polypeptide, the mRNA thatencodes and directs the synthesis of a subject polypeptide, etc. Alsoencompassed in this term are nucleic acids that are homologous,substantially similar or identical to the nucleic acids specificallydisclosed herein. A specific coding sequences of interest is:

Kamchatka Crab:

AAGCAGTGGTATCAACGCAGAGTACGCGGGGGAGATAGGACTGAGTGAGTGAGTGTGAGA (SEQ ID NO:02) GGGAAAGAAGGAATGGCCAACATGGAGTCCAAGCAAGGAATAATGGTTTTGGGATTCTTAATTGTCCTCCTCTTCGTGTCTGTCAATGGCCAGGACTGTGTGTGGGACAAGGACACGGACTTTCCCGAGGACCCGCCACTCATTTTCGATTCAAACTTGGAGCTCATCAGACCCGTCTTGGAAAATGGCAAAAGGATCGTCACTGTCCCCAGTGGCAGCAGCTTAACCTTGGCCTGCTCTGGGTCTGAACTGATCAACCTGGGCATGGAGGCGGTGGAAGCCAAGTGTGCTGGGGGAGTCATGCTTGCCATAGAAGGAACGGAGTGGGAGATCTGGAGCCTGGGGTGCAGCAACCACGTGAAGGAGACCATCCGCCGCAACCTTGGAACATGTGGGGAAGCGGACCAGGGGGATAGGCACAGTATTGGCTTCGAGTACTACGGTGGCTCCATCTATTATGAACTGATCAGCGTGTGTTTCGGGCCCGTGTCCGAAACAACCTTGCGCACCGAGCATGTCCTCCACGGCGCCAACATTGCCGCCAAGGACATCGAGACCTCCCGCCCCTCCTTCAAGACCTCCACCGGCTTCTTCAGCGTCTCCATGTCCACCGTCTACAGCCAGGCGTCACAGCTGCAGTTAATGACAGACATATTGGGAGATTCGGATCTAGCCAACAACATCATCGACCCCTCCCAACAGTTGTACTTCGCCAAAGGTCACATGTCTCCTGACGCAGACTTTGTGACGGTGGCAGAACAGGACGCCACCTACTACTTCATCAACGCCCTACCGCAGTGGCAGGCCTTCAATAATGGCAACTGGAAGTACCTAGAATACGCGACCCGAGACCTGGCCGAATCCCACGGTAGCGACCTGCGAGTGTATAGCGGAGGGTGGAGCCTGCTGCAGCTAGATGATATTAACGGCAACCCCGTGGACATCCTGCTGGGACTGTCTGAGGGCAAGGAGGTCGTGCCCGTTCCATCCCTCACTTGGAAGGTGGTTTACGAAGAGAGCAGCAGCAAAGCGCCCGCGATCGTCGGTATCAACAACCCTCACATCACCACCGCCCCCTCCCCCTTGTGTTCCGACCTGTGCTCCTCCTTGACCTGGATAGACTTCAACCTGGACGACCTGGCTCACGGGTACACCTACTGTTGTGCCGTAGATGACCTCCGGCAGGCCATACCCTATATCCCGGACCTGGGCAATGTCGGACTCCTCACTAACTAATTCTATCTATCTATCATATATGCTCAGGCCCAATCCCCATTTTGGGGGTAGCCGAACTCAAGAACAGAGCCAAGAAACAGGGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

Also provided are nucleic acids that are homologous to the providednucleic acid of SEQ ID NO:02, at least with respect to the coding regionthereof. The source of homologous nucleic acids to those specificallylisted above may be any species. In certain embodiments, the homologshave sequence similarity, e.g., at least 30% sequence identity, usuallyat least 40%, more usually at least 50% sequence identity betweennucleotide sequences. Sequence identity is calculated based on areference sequence, which may be a subset of a larger sequence, such asa conserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 nt long, more usually atleast about 30 nt long, and may extend to the complete sequence that isbeing compared. Algorithms for sequence analysis are known in the art,such as BLAST, described in Altschul etal. (1990), J. Mol. Biol.215:403-10 (using default settings, i.e. parameters w=4 and T=17).Unless indicated otherwise, the sequence similarity values reportedherein are those determined using the above referenced BLAST programusing default settings.

Of particular interest in certain embodiments are nucleic acidsincluding a sequence substantially similar to the specific nucleic acidsidentified above, where by substantially similar is meant havingsequence identity to this sequence of at least about 90%, usually atleast about 95% and more usually at least about 99%.

Also provided are nucleic acids that hybridize to the above describednucleic acids under stringent conditions. An example of stringenthybridization conditions is hybridization at 50° C. or higher and0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C. Stringenthybridization conditions are hybridization conditions that are at leastas stringent as the above representative conditions. Other stringenthybridization conditions are known in the art and may also be employedto identify nucleic acids of this particular embodiment of theinvention.

The subject nucleic acids may be cDNAs or genomic DNAs, as well asfragments thereof. The nucleic acids may also be mRNAs, e.g.,transcribed from genomic DNA, that encode (i.e. are translated into) thesubject proteins and polypeptides. Genomic DNA typically includes theopen reading frame encoding the subject proteins and polypeptides, andintrons, as well as adjacent 5′ and 3′ non-coding nucleotide sequencesinvolved, e.g., untranslated regions, promoter or other regulatoryelements, etc., in the regulation of expression, up to about 20 kbbeyond the coding region, but possibly further in either direction. Thegenomic DNA may be introduced into an appropriate vector forextrachromosomal maintenance or for integration into a host genome.

As such, a genomic sequence of interest comprises the nucleic acidpresent between the initiation codon and the stop codon, as defined inthe listed sequences, including all of the introns that are normallypresent in a native chromosome. It may further include specifictranscriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ and 3′ end of the transcribedregion. The genomic DNA may be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ or 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue and stage specific expression.

The term “cDNA” as used herein is intended to include all nucleic acidsthat share the arrangement of sequence elements found in native maturemRNA species, where sequence elements at least include exons. NormallymRNA species have contiguous exons, with the intervening introns, whenpresent, being removed by nuclear RNA splicing, to create a continuousopen reading frame encoding the subject proteins.

The nucleic acid compositions of the subject invention may encode all ora part of the subject proteins and polypeptides, described in greaterdetail above. Double or single stranded fragments may be obtained fromthe DNA sequence by chemically synthesizing oligonucleotides inaccordance with conventional methods, by restriction enzyme digestion,by PCR amplification, etc. For the most part, DNA fragments will be ofat least 15 nt, usually at least 18 nt or 25 nt, and may be at leastabout 50 nt.

The nucleic acids of the subject invention are isolated and obtained insubstantial purity, generally as other than an intact chromosome.Usually, the DNA will be obtained substantially free of other nucleicacid sequences that do not include a coding sequence or fragment thereoffor the subject proteins, generally being at least about 50%, usually atleast about 90% pure and are typically “recombinant,” i.e. flanked byone or more nucleotides with which it is not normally associated on anaturally occurring chromosome.

In addition to the plurality of uses described in greater detail infollowing sections, the subject nucleic acid compositions find use inthe preparation of all or a portion of the subjectproteins/polypeptides, as described below.

Also provided are nucleic acid probes, as well as constructs, e.g.,vectors, expression systems, etc., as described more fully below, thatinclude a nucleic acid sequence as described above. Probes of thesubject invention are generally fragments of the provided nucleic acid.The probes may be large or small fragments, generally ranging in lengthfrom about 10 to 100 or more nt, usually from about 15 to 50 nt. Inusing the subject probes, nucleic acids having sequence similarity aredetected by hybridization under low stringency conditions, for example,at 50° C. and 6×SSC (0.9 M sodium chloride/0.09 M sodium citrate)(oranalogous conditions) and remain bound when subjected to washing athigher stringency conditions, e.g., 55° C. in 1×SSC (0.15 M sodiumchloride/0.015 M sodium citrate) (or analogous conditions). Sequenceidentity may be determined by hybridization under stringent conditions,for example, at 50° C. or higher and 0.1×SSC (15 mM sodium chloride/01.5mM sodium citrate)(or analogous conditions). Nucleic acids having aregion of substantial identity to the provided nucleic acid sequencesbind to the provided sequences under stringent hybridization conditions.By using probes, particularly labeled probes of DNA sequences, one canisolate homologous or related sequences.

The subject nucleic acids are isolated and obtained in substantialpurity, generally as other than an intact chromosome. As such, they arepresent in other than their naturally occurring environment. Usually,the DNA will be obtained substantially free of other nucleic acidsequences that do not encode the above proteins/polypeptides, generallybeing at least about 50%, usually at least about 90% pure and aretypically “recombinant”, i.e. flanked by one or more nucleotides withwhich it is not normally associated on a naturally occurring chromosome.

The subject nucleic acids may be produced using any convenient protocol,including synthetic protocols, e.g., such as those where the nucleicacid is synthesized by a sequential monomeric approach (e.g., viaphosphoramidite chemistry); where subparts of the nucleic acid are sosynthesized and then assembled or concatamerized into the final nucleicacid, and the like. Where the nucleic acid of interest has a sequencethat occurs in nature, the nucleic acid may be retrieved, isolated,amplified etc., from a natural source using conventional molecularbiology protocols.

Also provided are constructs comprising the subject nucleic acidcompositions inserted into a vector, where such constructs may be usedfor a number of different applications, including propagation,polypeptide/protein production, and the like, as described in greaterdetail below. Constructs made up of viral and non-viral vector sequencesmay be prepared and used, including plasmids, as desired. The choice ofvector depends on the particular application in which the nucleic acidis to be employed. Certain vectors are useful for amplifying and makinglarge amounts of the desired DNA sequence. Other vectors are suitablefor expression in cells in culture, e.g., for use in screening assays.Still other vectors are suitable for transfer and expression in cells ina whole animal or person. The choice of appropriate vector is wellwithin the ability of those of ordinary skill in the art. Many suchvectors are available commercially. To prepare the constructs, thepartial or full-length nucleic acid is inserted into a vector typicallyby means of DNA ligase attachment to a cleaved restriction enzyme sitein the vector. Alternatively, the desired nucleotide sequence can beinserted by homologous recombination in vivo. Typically, homologousrecombination is accomplished by attaching regions of homology to thevector on the flanks of the desired nucleotide sequence. Regions ofhomology are added by ligation of oligonucleotides, or by polymerasechain reaction using primers that include both the region of homologyand a portion of the desired nucleotide sequence, for example.

Also provided are expression cassettes that include coding sequence ofthe subject nuclease. By expression cassette is meant a nucleic acidthat includes a sequence encoding a peptide or protein as describedabove operably linked to a promoter sequence, where by operably linkedis meant that expression of the coding sequence is under the control ofthe promoter sequence.

Preparation of the Subject Proteins

The subject proteins may be obtained using any convenient protocol. Assuch, they may be obtained from naturally occurring sources orrecombinantly produced. Naturally occurring sources of the subjectproteins include tissues and portions/fractions thereof, including cellsand fractions thereof, e.g., extracts, homogenates etc., that includecells in which the desired protein is expressed.

The subject proteins may also be obtained from synthetic protocols, e.g.by expressing a recombinant gene encoding the subject protein, such asthe polynucleotide compositions described above, in a suitable hostunder conditions sufficient for post-translational modification to occurin a manner that provides the expressed protein. For expression, anexpression cassette may be employed. The expression cassette or vectorwill provide a transcriptional and translational initiation region,which may be inducible or constitutive, where the coding region isoperably linked under the transcriptional control of the transcriptionalinitiation region, and under the translational control of thetranslational initiation region, and a transcriptional and translationaltermination region. These control regions may be native to the protein,or may be derived from exogenous sources.

Expression cassettes may be prepared comprising a transcriptioninitiation region, the nucleic acid coding sequence or fragment thereof,and a transcriptional termination region. Of particular interest is theuse of sequences that allow for the expression of functional epitopes ordomains, usually at least about 8 amino acids in length, more usually atleast about 15 amino acids in length, to about 25 amino acids, and up tothe complete open reading frame of the coding sequence. Afterintroduction of the DNA, the cells containing the construct may beselected by means of a selectable marker, the cells expanded and thenused for expression.

The subject proteins and polypeptides may be expressed in prokaryotes oreukaryotes in accordance with conventional ways, depending upon thepurpose for expression. For large scale production of the protein, aunicellular organism, such as E. coli, B. subtilis, S. cerevisiae,insect cells in combination with baculovirus vectors, or cells of ahigher organism such as vertebrates, particularly mammals, e.g. COS 7cells, may be used as the expression host cells. In some situations, itis desirable to express the coding sequence in eukaryotic cells, wherethe protein will benefit from native folding and post-translationalmodifications. Small peptides can also be synthesized in the laboratory.Polypeptides that are subsets of the complete protein sequence may beused to identify and investigate parts of the protein important forfunction.

Specific expression systems of interest include bacterial, yeast, insectcell and mammalian cell derived expression systems. Representativesystems from each of these categories is are provided below:

Bacteria. Expression systems in bacteria include those described inChang et al., Nature (1978) 275:615; Goeddel et al., Nature (1979)281:544; Goeddel et al., Nucleic Acids Res. (1980) 8:4057; EP 0 036,776;U.S. Pat. No. 4,551,433; DeBoer et al., Proc. Natl. Acad. Sci. (USA)(1983) 80:21-25; and Siebenlist et al., Cell (1980) 20:269.

Yeast. Expression systems in yeast include those described in Hinnen etal., Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et al., J.Bacteriol. (1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142;Kunze et al., J. Basic Microbiol. (1985) 25:141; Gleeson et al., J. Gen.Microbiol. (1986) 132:3459; Roggenkamp et al., Mol. Gen. Genet. (1986)202:302; Das et al., J. Bacteriol. (1984) 158:1165; De Louvencourt etal., J. Bacteriol. (1983) 154:737; Van den Berg et al., Bio/Technology(1990) 8:135; Kunze et al., J. Basic Microbiol. (1985) 25:141; Cregg etal., Mol. Cell. Biol. (1985) 5:3376; U.S. Pat. Nos. 4,837,148 and4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al., Curr.Genet. (1985) 10:380; Gaillardin et al., Curr. Genet. (1985) 10:49;Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284-289;Tilburn et al., Gene (1983) 26:205-221; Yelton et al., Proc. Natl. Acad.Sci. (USA) (1984) 81:1470-1474; Kelly and Hynes, EMBO J. (1985)4:475-479; EP 0 244,234; and WO 91/00357.

Insect Cells. Expression of heterologous genes in insects isaccomplished as described in U.S. Pat. No. 4,745,051; Friesen et al.,“The Regulation of Baculovirus Gene Expression”, in: The MolecularBiology Of Baculoviruses (1986) (W. Doerfler, ed.); EP 0 127,839; EP 0155,476; and Vlak et al., J. Gen. Virol. (1988) 69:765-776; Miller etal., Ann. Rev. Microbiol. (1988) 42:177; Carbonell et al., Gene (1988)73:409; Maeda et al., Nature (1985) 315:592-594; Lebacq-Verheyden etal., Mol. Cell. Biol. (1988) 8:3129; Smith et al., Proc. Natl. Acad.Sci. (USA) (1985) 82:8844; Miyajima et al., Gene (1987) 58:273; andMartin et al., DNA (1988) 7:99. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts aredescribed in Luckow et al., Bio/Technology (1988) 6:47-55, Miller etal., Generic Engineering (1986) 8:277-279, and Maeda et al., Nature(1985) 315:592-594.

Mammalian Cells. Mammalian expression is accomplished as described inDijkema et al., EMBO J. (1985) 4:761, Gorman et al., Proc. Natl. Acad.Sci. (USA) (1982) 79:6777, Boshart et al., Cell (1985) 41:521 and U.S.Pat. No. 4,399,216. Other features of mammalian expression arefacilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:44,Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat. Nos.4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO 87/00195,and U.S. RE 30,985.

When any of the above host cells, or other appropriate host cells ororganisms, are used to replicate and/or express the polynucleotides ornucleic acids of the invention, the resulting replicated nucleic acid,RNA, expressed protein or polypeptide, is within the scope of theinvention as a product of the host cell or organism.

Once the source of the protein is identified and/or prepared, e.g. atransfected host expressing the protein is prepared, the protein is thenpurified to produce the desired protein comprising composition. Anyconvenient protein purification procedures may be employed, wheresuitable protein purification methodologies are described in Guide toProtein Purification, (Deuthser ed.) (Academic Press, 1990). Forexample, a lysate may be prepared from the original source, e.g.naturally occurring cells or tissues that express the protein or theexpression host expressing the protein, and purified using HPLC,exclusion chromatography, gel electrophoresis, affinity chromatography,and the like.

Also of interest is the use of modified versions of the wild typesequences which are modified to provide for optimized expression in aparticularly type of expression host. For example, humanized versions ofthe subject nucleic acids can be used for expression in human celllines, where changes are made to the wild type nucleic acid sequence tooptimize the codons for expression of the protein in human cells (Yanget al., Nucleic Acids Research 24 (1996), 4592-4593). See also U.S. Pat.No. 5,795,737 which describes humanization of proteins, the disclosureof which is herein incorporated by reference.

In certain embodiments, the subject proteins are produced as fusionproteins. In these embodiments, nucleic acids that encode fusionproteins of the subject proteins, or fragments thereof, which are fusedto a second protein, a tagging sequence, etc. Fusion proteins maycomprise a subject polypeptide, or fragment thereof, and a DNasepolypeptide (“the fusion partner”) fused in-frame at the N-terminusand/or C-terminus of the subject polypeptide. Fusion partners include,but are not limited to, polypeptides that can bind antibody specific tothe fusion partner (e.g., epitope tags); antibodies or binding fragmentsthereof; polypeptides that provide a catalytic function or induce acellular response; ligands or receptors or mimetics thereof; and thelike. In such fusion proteins, the fusion partner is generally notnaturally associated with the subject nuclease portion of the fusionprotein, and is typically not an nuclease protein or derivative/fragmentthereof. Of particular interest in many protein production applicationis the use of fusion partners encoding metal ion peptide affinity tags,e.g., the 6×His tag and other metal ion affinity tags, where the tagsprovide for ready purification on appropriate metal ion affinity resins.Metal ion affinity tagged peptide technology is well known to those ofskill in the art, and is described in U.S. Pat. Nos. 5,284,933;5,310,663; 4,569,794; 5,594,115 and 6,242,581; the disclosures of whichare herein incorporated in their entirety.

The subject polypeptides, peptides, variants and or fragments thereofmay also be prepared through chemical synthesis. The polypeptides may bemonomers or multimers; glycosylated or non-glycosylated; pegylated ornon-pegylated; amidated or non-amidated; sulfated or non-sulfated; andmay or may not include an initial methionine amino acid residue. Forexample, the polypeptides can also be synthesized by exclusive solidphase synthesis, partial solid phase methods, fragment condensation orclassical solution synthesis. The polypeptides are in many embodimentsprepared by solid phase peptide synthesis, for example as described byMerrifield, J. Am. Chem. Soc. 85:2149, 1963. The synthesis is carriedout with amino acids that are protected at the alpha-amino terminus.Trifunctional amino acids with labile side-chains are also protectedwith suitable groups to prevent undesired chemical reactions fromoccurring during the assembly of the polypeptides. The alpha-aminoprotecting group is selectively removed to allow subsequent reaction totake place at the amino-terminus. The conditions for the removal of thealpha-amino protecting group do not remove the side-chain protectinggroups.

The alpha-amino protecting groups are those known to be useful in theart of stepwise polypeptide synthesis. Included are acyl type protectinggroups (e.g., formyl, trifluoroacetyl, acetyl), aryl type protectinggroups (e.g., biotinyl), aromatic urethane type protecting groups [e.g.,benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl and9-fluorenylmethyloxy-carbonyl (Fmoc)], aliphatic urethane protectinggroups [e.g., t-butyloxycarbonyl (tBoc), isopropyloxycarbonyl,cyclohexioxycarbonyl] and alkyl type protecting groups (e.g., benzyl,triphenylmethyl). The preferred protecting groups are tBoc and Fmoc.

The side-chain protecting groups selected must remain intact duringcoupling and not be removed during the deprotection of theamino-terminus protecting group or during coupling conditions. Theside-chain protecting groups must also be removable upon the completionof synthesis using reaction conditions that will not alter the finishedpolypeptide. In tBoc chemistry, the side-chain protecting groups fortrifunctional amino acids are mostly benzyl based. In Fmoc chemistry,they are mostly tert-butyl or trityl based.

In tBoc chemistry, the preferred side-chain protecting groups are tosylfor arginine, cyclohexyl for aspartic acid, 4-methylbenzyl (andacetamidomethyl) for cysteine, benzyl for glutamic acid, serine andthreonine, benzyloxymethyl (and dinitrophenyl) for histidine,2-CI-benzyloxycarbonyl for lysine, formyl for tryptophan and2-bromobenzyl for tyrosine. In Fmoc chemistry, the preferred side-chainprotecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) or2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine,trityl for asparagine, cysteine, glutamine and histidine, tert-butyl foraspartic acid, glutamic acid, serine, threonine and tyrosine, tBoc forlysine and tryptophan.

For the synthesis of phosphopeptides, either direct or post-assemblyincorporation of the phosphate group is used. In the directincorporation strategy, the phosphate group on serine, threonine ortyrosine may be protected by methyl, benzyl, or tert-butyl in Fmocchemistry or by methyl, benzyl or phenyl in tBoc chemistry. Directincorporation of phosphotyrosine without phosphate protection can alsobe used in Fmoc chemistry. In the post-assembly incorporation strategy,the unprotected hydroxyl groups of serine, threonine or tyrosine arederivatized on solid phase with di-tert-butyl-, dibenzyl- ordimethyl-N,N′-diisopropylphosphoramidite and then oxidized bytert-butylhydroperoxide.

Solid phase synthesis is usually carried out from the carboxyl-terminusby coupling the alpha-amino protected (side-chain protected) amino acidto a suitable solid support. An ester linkage is formed when theattachment is made to a chloromethyl, chlortrityl or hydroxymethylresin, and the resulting polypeptide will have a free carboxyl group atthe C-terminus. Alternatively, when an amide resin such asbenzhydrylamine or p-methylbenzhydrylamine resin (for tBoc chemistry)and Rink amide or PAL resin (for Fmoc chemistry) are used, an amide bondis formed and the resulting polypeptide will have a carboxamide group atthe C-terminus. These resins, whether polystyrene- or polyamide-based orpolyethyleneglycol-grafted, with or without a handle or linker, with orwithout the first amino acid attached, are commercially available, andtheir preparations have been described by Stewart et al., “Solid PhasePeptide Synthesis” (2nd Edition), (Pierce Chemical Co., Rockford, Ill.,1984) and Bayer & Rapp Chem. Pept. Prot. 3:3 (1986); and Atherton etal., Solid Phase Peptide Synthesis: A Practical Approach, IRL Press,Oxford, 1989.

The C-terminal amino acid, protected at the side chain if necessary, andat the alpha-amino group, is attached to a hydroxylmethyl resin usingvarious activating agents including dicyclohexylcarbodiimide (DCC),N,N′-diisopropylcarbodiimide (DIPCDI) and carbonyldiimidazole (CDI). Itcan be attached to chloromethyl or chlorotrityl resin directly in itscesium tetramethylammonium salt form or in the presence of triethylamine(TEA) or diisopropylethylamine (DIEA). First amino acid attachment to anamide resin is the same as amide bond formation during couplingreactions.

Following the attachment to the resin support, the alpha-aminoprotecting group is removed using various reagents depending on theprotecting chemistry (e.g., tBoc, Fmoc). The extent of Fmoc removal canbe monitored at 300-320 nm or by a conductivity cell. After removal ofthe alpha-amino protecting group, the remaining protected amino acidsare coupled stepwise in the required order to obtain the desiredsequence.

Various activating agents can be used for the coupling reactionsincluding DCC, DIPCDI, 2-chloro-1,3-dimethylimidium hexafluorophosphate(CIP), benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluoro-phosphate (BOP) and its pyrrolidine analog (PyBOP),bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP),O-(benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate(HBTU) and its tetrafluoroborateanalog (TBTU) or its pyrrolidine analog(HBPyU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumhexafluorophosphate (HATU) and its tetrafluoroborate analog (TATU) orits pyrrolidine analog (HAPyU). The most common catalytic additives usedin coupling reactions include 4-dimethylaminopyridine (DMAP),3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt),N-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole (HOAt).Each protected amino acid is used in excess (>2.0 equivalents), and thecouplings are usually carried out in N-methylpyrrolidone (NMP) or inDMF, CH₂Cl₂ or mixtures thereof. The extent of completion of thecoupling reaction can be monitored at each stage, e.g., by the ninhydrinreaction as described by Kaiser et al., Anal. Biochem. 34:595, 1970.After the entire assembly of the desired peptide, the peptide-resin iscleaved with a reagent with proper scavengers. The Fmoc peptides areusually cleaved and deprotected by TFA with scavengers (e.g., H₂O,ethanedithiol, phenol and thioanisole). The tBoc peptides are usuallycleaved and deprotected with liquid HF for 1-2 hours at −5 to 0° C.,which cleaves the polypeptide from the resin and removes most of theside-chain protecting groups. Scavengers such as anisole,dimethylsulfide and p-thiocresol are usually used with the liquid HF toprevent cations formed during the cleavage from alkylating and acylatingthe amino acid residues present in the polypeptide. The formyl group oftryptophan and the dinitrophenyl group of histidine need to be removed,respectively by piperidine and thiophenyl in DMF prior to the HFcleavage. The acetamidomethyl group of cysteine can be removed bymercury(II)acetate and alternatively by iodine,thallium(III)trifluoroacetate or silver tetrafluoroborate whichsimultaneously oxidize cysteine to cystine. Other strong acids used fortBoc peptide cleavage and deprotection include trifluoromethanesulfonicacid (TFMSA) and trimethylsilyltrifluoroacetate (TMSOTf).

Antibodies

Also provided are antibodies that bind to the subject proteins and/orhomologs thereof. Suitable antibodies are obtained by immunizing a hostanimal with peptides comprising all or a portion of the subject protein.Suitable host animals include rat, sheep, goat, hamster, rabbit, etc.The host animal will generally be a different species than theimmunogen, e.g. human used to immunize rabbit, etc.

The immunogen may comprise the complete protein, or fragments andderivatives thereof. Preferred immunogens comprise all or a part of thesubject protein, where these residues contain the post-translationmodifications, such as glycosylation, found on the native target protein(immunogens may also comprise all or a part of the subject protein,where these residues does not contain the post-translationmodifications).

Immunogens comprising the extracellular domain are produced in a varietyof ways known in the art, e.g. expression of cloned genes usingconventional recombinant methods, isolation from naturally occurringsources, etc.

For preparation of polyclonal antibodies, the first step is immunizationof the host animal with the target protein, where the target proteinwill preferably be in substantially pure form, comprising less thanabout 1% contaminant. The immunogen may include the complete targetprotein, fragments or derivatives thereof. To increase the immuneresponse of the host animal, the target protein may be combined with anadjuvant, where suitable adjuvants include alum, dextran, sulfate, largepolymeric anions, oil & water emulsions, e.g. Freund's adjuvant,Freund's complete adjuvant, and the like. The target protein may also beconjugated to synthetic carrier proteins or synthetic antigens. Avariety of hosts may be immunized to produce the polyclonal antibodies.Such hosts include rabbits, guinea pigs, rodents, e.g. mice, rats,sheep, goats, and the like. The target protein is administered to thehost, usually intradermally, with an initial dosage followed by one ormore, usually at least two, additional booster dosages. Followingimmunization, the blood from the host will be collected, followed byseparation of the serum from the blood cells. The Ig present in theresultant antiserum may be further fractionated using known methods,such as ammonium salt fractionation, DEAE chromatography, and the like.

Monoclonal antibodies of the subject invention may be produced byconventional techniques. Generally, the spleen and/or lymph nodes of animmunized host animal provide a source of plasma cells. The plasma cellsare immortalized by fusion with myeloma cells to produce hybridomacells. Culture supernatant from individual hybridomas is screened usingstandard techniques to identify those producing antibodies with thedesired specificity. Suitable animals for production of monoclonalantibodies to the human protein include mouse, rat, hamster, etc. Toraise antibodies against the mouse protein, the animal will generally bea hamster, guinea pig, rabbit, etc. The antibody may be purified fromthe hybridoma cell supernatants or ascites fluid by conventionaltechniques, e.g. affinity chromatography using MPTS bound to aninsoluble support, protein A sepharose, etc.

The antibody may be produced as a single chain, instead of the normalmultimeric structure. Single chain antibodies are described in Jost etal. (1994) J.B.C. 269:26267-73, and others. DNA sequences encoding thevariable region of the heavy chain and the variable region of the lightchain are ligated to a spacer encoding at least about 4 amino acids ofsmall neutral amino acids, including glycine and/or serine. The proteinencoded by this fusion allows assembly of a functional variable regionthat retains the specificity and affinity of the original antibody.

The antibodies or fragments thereof may also be produced using phagedisplay technology. Phage display technology is well known to those ofskill in the art, where representative patents describing variousrepresentative embodiments of this technology include U.S. Pat. Nos.5,427,908; 5,580,717; 5,658,727; 5,723,287; 5,750,373; 5,780,279;5,821,047; 5,846,765; 5,885,793; 5,955,341; 6,040,136; 6,057,098; and6,172,197; the disclosures of which are herein incorporated byreference.

Kits

Also provided are kits for use in practicing the subject methods. Themethods of the invention can be facilitated by the use of kits thatcontain the reagents required for carrying out the assays. The kits cancontain reagents for carrying out the analysis of a one singlenucleotide polymorphism (SNP) site (for use in, e.g., diagnosticmethods) or multiple SNP sites (for use in, e.g., genomic mapping). Whenmultiple samples are analyzed, multiple sets of the appropriate primersand oligonucleotides are provided in the kit. In addition to the primersand oligonucleotides required for carrying out the various methods, thekits contain the nuclease enzyme, such as the novel nucleases describedabove, and DSN buffer (e.g., buffer where these nucleases workcorrectly). Also the kits may contain the reagents for detecting thelabels and for nucleic acids amplification. The kits can also containsolid substrates for use in carrying out the method of the invention.For example, the kits can contain solid substrates, such as glass platesor silicon or glass microchips, containing arrays of nucleic acidprobes.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internet to access the information at a removed site. Anyconvenient means may be present in the kits.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

I. Isolation and Characterization of Kamchatka Crab nuclease

A. Purification of Crab Nuclease Protein

A specific nuclease activity was found in crab hepatopancreas. Theactivity was purified by standard methods of protein chemistry. Becausethe specific activity of this enzyme is thermostable, both purified andpartially purified samples (after warming) of crab nuclease display thespecific activity.

The purification scheme may be different and may include differentstages. A representative scheme is provided below:

Crab DSN nuclease was purified at 4° C. Fractions were subjected totesting for DNase activity and Western blotting (Harlow and Lane, 1988)with rabbit polyclonal antibodies against recombinant protein.

Fresh crab hepatopancreas was homogenized in two volumes of 100 mMTris-HCl (pH 8.0) with 100 mM EDTA and centrifuged at 10 000 g for 30min. The supernatant was diluted with 1.5 volumes of acetone, incubatedfor 12 h and centrifuged at 10 000 g for 1 h. The sediment was dried and5 g of the resultant acetone powder was diluted in 250 ml buffer A (0.05M Tris-HCl, pH 7.1) and mixed for 2 h. The insoluble fraction wasseparated out by centrifugation at 10 000×g for 30 min, and thesupernatant was applied to a DEAE-MacroPrep column (Bio-Rad)equilibrated with buffer A. After loading, the column was washed withthe same buffer. Protein was eluted with a 0-0.5 M NaCl gradient inbuffer A. After adding NaCl to a final concentration of 5 M,DSN-containing fractions were loaded onto a Phenyl-Agarose column(Amersham-Pharmacia-Biotech) equilibrated with buffer A containing 5 MNaCl. After washing the column with the same high-salt buffer, DSN waseluted with a 5-3 M NaCl gradient in buffer A. DSN-containing fractionswere pooled, diluted two-fold with 0.035 M Tris-HCl (pH 8.1) and appliedto a Hydroxyapatite column (Bio-Rad) equilibrated with 0.045 M Tris-HCl(pH 7.5). The fractions were directly eluted with 0.025 M sodiumphosphate buffer, pH 7.5, combined and dialyzed overnight against 0.01 MTris-HCl (pH 7.1) containing 0.001 M MgCl₂. Dialyzed fractions wereloaded onto a Heparin-Sepharose column (Amersham-Pharmacia-Biotech),equilibrated with the 0.01 M Tris-HCl (pH 7.1) buffer containing 0.001 MMgCl₂, and eluted with a 0-0.3 M NaCl gradient after washing.DSN-containing fractions were concentrated to 1 ml on a Biomax-5Kmembrane (Millipore), transferred to buffer A and subjected to gelfiltration on a Sephadex G-75 (Sigma) column. Purified DSN wasconcentrated on Biomax-5K membrane, diluted with one volume of glycerol,heated at 70° C. for 10 min under mineral oil and incubated at +4° C.overnight. About 0.15 mg DSN protein (2700 Kunitz units) was purifiedfrom 5 g acetone powder. DSN was stored at −20° C. for long-term use.

B. Isolation of the Crab Nuclease Coding Sequence.

To isolate coding sequence of crab nuclease and its homologues fromother species, a set of oligonucleotide primers were designed to theconserved amino acids of the various nucleases: Serratia marcescensnuclease (1583130), Glossina morsitans nuclease (AAF82097), Homo sapiensnuclease (XP_(—)002889), Syncephalastrum racemosum nuclease (P81204),Cunninghamella echinulata nuclease (P81203), Drosophila melanogasternuclease (AAF49206) and Penaeus japonicus nuclease (CAB55635).

NN Primer name Primer Sequence 1. PQW1 5′-TAC ATT AAT GCC GTC CCT CAGTGG-3′ (SEQ ID NO:03) 2. GNW1 5′-CAG GCC TTT AAT AAT GGT AAT TGG-3′ (SEQID NO:04) 3. GNW2 5′-AG GCC TTT AAT AA(T/C) GGT AAC TGG-3′ (SEQ IDNO:05) 4. AFN1 5′-CCT CAG TGG CA(G/A) GCT TT(C/T) AAT-3′ (SEQ ID NO:06)5. AFN2 5′-CCT CAG TGG CA(G/A) GCT TT(C/T) AAC-3′ (SEQ ID NO:07)

All primers were purified through polyacrylamide gel before use.

For crab nuclease cDNA isolation, the modified method for amplifyingcDNA ends based on SMART-RACE technology (Clontech Laboratories Inc.,Palo Alto, Calif.) was used. SMART-RACE technology also noted asstep-out RACE has been described in (Matz, M., Shagin, D., Bogdanova,E., Britanova, O., Lukyanov, S., Diatchenko, L., Chenchik, A. (1999)Nucleic Acids Res. 27, 1558-1560). Total RNA from King crabhepatopancreas was isolated as described in (Chomczynski, P., Sacchi, N.(1987) Anal. Biochem. 162, 156-159). The cDNA was amplified by a SMARTPCR cDNA Synthesis Kit (CLONTECH) using the provided protocol and thenused for 3′-step-out RACE (Matz et al., 1999). PCR reaction wasperformed in a 25 mkl reaction mixture containing 1 mkl of 20-foldamplified cDNA, 1× Advantage 2 Polymerize mix (CLONTECH), themanufacturer's 1× reaction buffer, 200 mkM dNTPs, 0.3 mkM of AFN1degenerative primer, and step-out primer system (0.02 mkM of“heel-carrier” oligo and 0.15 mkM of “heel-specific” oligo). 30 PCRcycles were performed in PTC-200 MJ Recearch Termal Cycler in calculatedcontrol mode (each cycle included 95° C.-10 s; 60° C.-10 s; 72° C.-2min). 1/1000 of resulted amplified cDNA was used for nested PCR withGNW1 primer. PCR reaction was performed in a 25 mkl reaction mixturecontaining 1× Advantage 2 Polymerize mix (CLONTECH), the manufacturer's1× reaction buffer, 200 mkM dNTPs, 0.3 mkM of GNW1 primer, and step-outprimer system (0.02 mkM of “heel-carrier” oligo and 0.15 mkM of“heel-specific” oligo). 21 PCR cycles were performed in PTC-200 MJRecearch Termal Cycler in calculated control mode (each cycle included95° C.-10 s; 64° C.-10 s; 72° C.-2 min).

PCR products were cloned in pTAdv-cloning vector (CLONTECH) andsequenced using M13 direct and reverse universal primer by using aBeckman SEQ-2000 automated sequencer and the FS dye terminatorchemistry.

cDNA containing the full coding sequence of crab DSN was then isolatedusing SMART-RACE technology (Clontech Laboratories Inc., Palo Alto,Calif.). For additional RACE procedures the crab nuclease specificprimers were used as following: 5′-GGC CAG GTC TCG GGT CGC-3′; 5′-GGGTCG CGT ATT CTA GGT A-3′ (SEQ ID NO:08); 5′-CC ATT ATT GAA GGC CTGCCA-3′ (SEQ ID NO:09).

The nt sequence of the isolated cDNA is provided in SEQ ID NO:01 and theamino acid sequence of the protein encoded thereby is provided in SEQ IDNO:02, which sequences are provided above.

C. Generation of Antibodies

Polyclonal antibodies were prepared to His-tagged mature DSN proteinproduced in Escherichia coli. Recombinant products were purified byimmobilized metal affinity chromatography using Talon Resin (ClontechLaboratories, Inc.) under denaturing conditions. Rabbits were immunizedand boosted four times at monthly intervals with recombinant DSNpolypeptide emulsified in complete Freund's adjuvant. Ten or 11 daysafter each boost the animals were bled. Polyclonal antiserum was testedon recombinant protein by ELISA and by Western immunobloting. Polyclonalantiserum was then used for Western immunobloting with total proteinextract from crab hepatopancreas and partially purified DSN samples. Thesamples enriched in specific activity had greater staining thanunpurified samples.

D. Testing of DSN Activity.

The above cloned crab Double stranded nuclease (DSN) is a Mg²⁺—dependentnuclease with following properties:

-   -   DNAse activity of the subject nuclease is at least 18 000        Kunitz-units per 1 mg of the protein as determined by modified        DNase activity assay (Kunitz M, 1950, J. Gen. Physiol. 33, pages        349-362; Liao, T.-H. 1974 J. Biol. Chem. 249: pages 2354-2356).        Activity was determined in reaction mixture containing 5 mM        Tris-HCl, pH 7.5, 10 mM MgCl₂, 10 mM CaCl₂, 400 mkg calf thymus        DNA and crab nuclease in different concentration. Nuclease        concentration was determined using Bradford method (Bradford M.,        Anal. Biochem., 1976, v. 72, p. 248-254).    -   In the absence of Mg²⁺ ions crab DSN is inactive.    -   In the presence of Mg²⁺-ions and in the absence of Ca²⁺-ions,        only of about 50% of nuclease activity keeps as was measured by        modified Kunitz assay.    -   crab DSN exhibited strong cleavage preference for ds DNA        substrates (DNA-DNA and DNA in DNA-RNA hybrids) and little        activity against ss DNA. No significant cleavage activity on RNA        substrates was observed (RNase activity was measured essentially        as described by Ho H C., Shiau P. F., Liu F. C., Chung J. G.,        Chen L. Y. Eur J Biochem. 1998, 256: 112-118).

Following examples demonstrate structure-specificity of crab DSNdescribed above:

-   -   1. DSN activity on λ ds DNA and phage M13 ss-DNA was compared by        agarose gel electrophoresis. The reaction was performed in a        total volume of 10 μl comprising 1×DSN buffer (7 mM MgCl₂, 50 mM        Tris-HCl, pH 8.0), 0.06 Kunitz/units DSN, 150 ng λ DNA and 50 ng        M13 DNA. To prevent ds structure formation in phage M13 DNA, the        reaction mixture was incubated at 70° C. for 1, 5 or 5 min. The        digestion products were visualized on a 0.9% agarose gel,        following ethidium bromide staining (FIG. 2).    -   2. For analysis of crab DSN activity on synthetic        oligonucleotide substrates, oligonucleotides labeled with a        fluorescent donor at the 5′ end and a fluorescent quencher at        the 3′ end were used as ss DNA. To generate ds substrates,        labeled oligonucleotides were mixed with equimolar amounts of        complementary non-labeled oligonucleotides.        -   2.1. Action of crab nuclease on synthetic ss and ds 20-mer            DNA substrates was performed in a total volume of 20 μl            comprising 1×DSN buffer 50 mM Tris-HCl, pH 8.0 and 7 mM            MgCl₂), 0.6 Kunitz units DSN, and 0.3 μM oligonucleotide            substrate. Incubation was carried out at 35° C. for            different periods. DNase activity was evaluated by            estimating the change in fluorescence intensity of the            reaction mixture during incubation with DSN. Fluorescence            intensity was measured on a spectrofluorimeter Cary Eclypse            (Varian) in 2 ml dishes (FIG. 3).        -   2.2. Action of crab nuclease on synthetic ds DNA substrates            of different length was performed in a total volume of 20 μl            comprising 1×DSN buffer, 1.5 Kunitz units DSN, and 0.3 μM            oligonucleotide substrate. Incubation was carried out at            35° C. for different periods. DNase activity was evaluated            by estimating the change in fluorescence intensity of the            reaction mixture during incubation with DSN. Fluorescence            intensity was measured on a spectrofluorimeter Cary Eclypse            (Varian) in 2 ml dishes (FIG. 4). Cleavage curves were            plotted to obtain half-time for substrate cleavage (T1/2).    -   DSN is significantly more effective in cutting perfect DNA-DNA        and DNA-RNA duplexes than it is in cutting non-perfect duplexes        of the same length. The following example demonstrates the        structure-specificity of crab DSN described above: To determine        the generality of mismatch discrimination, we constructed a set        of closely related 18 nt synthetic targets with single        nucleotide variations along the 10 nt sequence and FRET-labeled        10 nt probe oligonucleotide capable of hybridizing with these        targets to form perfect and one mismatch-containing duplexes.        Two types of signal probes were used, specifically, probes        labeled with 5-carboxyfluorescein (Fl) (at the 5′ end) and TAMRA        (at the 3′ end) and those labeled with Fl and DABCYL. Duplexes        formed by probe oligonucleotides and complementary targets were        incubated with DSN at 35° C. for 15 min. Reactions were        performed in a total volume of 20 μl comprising 1×DSN buffer,        0.6 Kunitz units DSN, and 0.3 μM oligonucleotide substrate.        Emission spectra were obtained on the spectrofluorimeter, with        excitation at 480 nm (see FIG. 5 for example). All possible        combinations between targets and probe oligonucleotides were        examined for cleavage by DSN. The observed fluorescence change        for all mismatched duplexes was at least 10 times lower than        that for perfectly matched duplexes in the experiments with        Fl-TAMRA-labeled probe. No preference of DSN for specific        mismatch positions was noted. In the case of the Fl-DABCYL        labeled probe, clear discrimination between perfect and        non-perfect duplexes was observed if the variable nucleotide        position was either at the 5′ end or the center of a signal        probe. Duplexes comprising mismatches near the 3′ end of the        signal probe (T1-4) were cleaved by DSN with only 1.5-5 times        less efficiency than perfect duplexes.

Examples that demonstrate the specific activity of crab DSN on perfectlyand non-perfectly matched DNA containing duplexes are also shown in“Examples of DSNP ASSAY Methods” section (below).

-   -   The pH and temperature optima for crab DSN activity are 7-8 and        55-65° C., respectively. The nuclease is stable at a pH of        greater than 6, and temperatures below 75° C. The dependence of        DSN activity on pH was analyzed using the Kunitz assay with 50        mM sodium formiate (pH 3.0-3.5), 50 mM sodium acetate (pH        3.5-6.0), 50 mM Mes-NaOH (pH 6.0-7.0), 50 mM Tris-HCl (pH        7.0-9.5) and 50 mM glycine-NaOH (pH 9.0-10.0) in the presence of        7 mM MgCl₂. The dependence of DSN activity on temperature was        analyzed at different reaction temperatures, ranging between 20        and 90° C. The activity of crab DSN in different temperatures is        demonstrated graphically in FIG. 6. The activity of crab DSN in        different Mg²⁺ concentration is demonstrated graphically in FIG.        7.    -   Crab DSN is inactive in the absence of divalent cations. EDTA        inhibits the Crab DSN. Crab DSN is also fully inactivated by        heating at temperature 97° C. (or higher) for 7-10 min.        E. Cloning of Putative Nucleases of DSN Family from Other        Arthropoda Species

Using a modified method for amplifying cDNA ends based on SMART-RACEtechnology and primers described above, several cDNAs that encodeproteins homologues to crab DSN have been cloned. Amplificationreactions contained 1× Advantage 2 Polymerize mix (CLONTECH), themanufacturer's 1× reaction buffer, 200 mkM dNTPs, 0.3 mkM of primer, andstep-out primer system (0.02 mkM of “heel-carrier” oligo and 0.15 mkM of“heel-specific” oligo) in a volume of 25 μl. PCR was carried out in 200MJ Recearch Termal Cycler in calculated control mode.

For isolation of the gammarus (Gammarus sp.) putative nuclease 34 cyclesPCR were performed with AFN2 primer by the following program: 95° C.-10s; 62° C.-10s; 72° C.-2 min.

For isolation of the glass shrimp (Palaemonidae sp) and Mangrove FiddlerCrab (Uca crassipes) putative nucleases two consistent PCRs were carriedout. The AFN2 primer was used for the first PCR that included 32 cyclesby the following program: 95° C.-10 s; 60° C.-10 s; 72° C.-2 min and theGNW1 primer was used for the second (nested) PCR that included 25 cycles(each cycle included 95° C.-10 s; 64° C.-10 s; 72° C.-2 min).

cDNAs containing the full coding sequences of these nucleases were thenisolated using SMART-RACE technology (Clontech Laboratories Inc., PaloAlto, Calif.) with following gene-specific primers:

Fiddler Crab nuclease-specific primers: 5′-GG ATT GCC ATT AAT GTC GTC-3′(SEQ ID NO:10); 5′-CC ACT GTA CAC CCG AAG GTC-3′ (SEQ ID NO:11); 5′-AACCAA GGC TCG CCA AGT CC-3′ (SEQ ID NO:12); gammarus nuclease-specificprimers: 5′-C AAT GGT CCG AAT TCT GTT CTC-3′ (SEQ ID NO:13); 5′-GTG ACTACG CGC AGA GTG GC-3′ (SEQ ID NO:14); and glass shrimp nuclease-specificprimers: 5′-CCA GCA CTC CCC AAC CTC C-3′ (SEQ ID NO:15); 5′-GTC AGG TCAGTG CCG TGG GC-3′ (SEQ ID NO:16).

The nt sequences of the isolated cDNA are provided in SEQ ID NO:17; SEQID NO:19; and SEQ ID NO:21 and the amino acid sequences of the proteinencoded thereby are provided in SEQ ID NO:18; SEQ ID NO:20; and SEQ IDNO:22, which sequences are provided below:

Gammarus Putative Nuclease:

Source: Eukaryota; Metazoa; Arthropoda; Crustacea; Malacostraca;Eumalacostraca; Peracarida; Amphipoda; Gammaridea; Gammaroidea;Gammaridae; Gammarus; Gammarus sp.

Nucleic Acid Composition

TGTAGCGTGAAGACGACAGAAGTAATACGACTCACTATAGGGCAAGCAGTG (SEQ ID NO: 17)GTACCAACGCAGAGTACTCGTCTTCCTTTTTTTTTTTGGTGTTGCTGCCCCACTGTGCAAACTTGAGGCGCTGCTCTTGGCAACTGAGAAATTCTACGAATCAGACGATACACGCTTCATAGTTCAGACCACTTTTTGTTGTACAACTATTTTTTTTTATTATTATTTTAGTTGTATTTTATTATTTGAAGAGAAGAAAGTTTGTGCTACAATTTCAACACTGCTTCACAATATTGTCTTATACAGGCAATTGGGAAATACATCACTAGAGCGAGACTCCGAATATCTGTCACTTCAAAAGCCGGTCGAGAACTTCGAATGCGAGAGCTCCATCCAGTACAATGTTTTGCCCTTGGGAGGAGGAAACGAAGAGGACGAGGAGGATAGTATTGGAGGGGTGAGCGCTTTTGGCTATGACCTCAAGGAAACTTTCTACACCATCTACAAGTTTGAGTTTGATAAGACGAAGATGATGAGCAGCCGAGTGGACCACATCCTCCACGGCAGGGAGTTATTGAAGGCCGAGCCTCACAGGGATTACAAGTTCAGAAGTGATGCAGTCCTCCCTTGGGGAAGCCTCACCAAGATCAAGAAATGCTACTCGAATCAGAATCAAGATGCGGTGCTGCAGCACTTCCAAGATCAGGGAAGTAATGAGAAAAGAAAATACTTTGCGCGAGGTCACCTGGCAGCAAATGCAGACTTCGTGTCACAAGACGAGCAGAAAGCTTCCTACAGCTTCGCCAACGTAGCGCCTCAGTGGCAGGCTTTCAACAATGGCAACTGGAAGAAACTTGAGAACAGAATTCGGACCATTGCCATGAAAAAGGAAGCCACTCTGCGCGTAGTCACTGGCACCTCCACGGGTCTTCTGAAGCTCAATAGTAGTTCTGGACAACTGGAGAACGTGTGCCTGTGTGACGGTGAGCCGCCGTGTGTTCCTCTCTTCTTTTGGAAGGTGACGCCCGCGTTAAGACAAGCGTTCCTGATGCCTAATCATCCTGCCGCGGGAATATCAGCCAGAATTGAAGAACTGGCACATCCTTGTCCGAATTGGGCAGGTTTCTCTGGAGCCGAAGAGAAGACTCGAGGAGCCCTGTATTGCCTCACCCCAGGCGACCTCTGTGGAGTTGAGCCGAAGGCTTGTGGATACAAAGATTTAATGTGAACTCTATATTTGCATTTGAATAACCTTCGTACGTCATTTGGTCAAATTTTGCTCTGTGTTTGTATATACAAAATGTCCAGGAATGGCCAAAAGTGCAGAGGTTGGTAAATTCTTAGAGTTCAACCTCGAAGTTGATACTAATAGTTTTGAAAATTGAAATATATGTTTAACGCAGTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAmino Acid Composition

GKQWYQRRVLVFLFFFGVAAPLCKLEALLLATEKFYESDDTRFIVQTTFCCTTIF (SEQ ID NO: 18)FYYYFSCILLFEEKKVCATISTLLHNIVLYRQLGNTSLERDSEYLSLQKPVENFECESSIQYNVLPLGGGNEEDEEDSIGGVSAFGYDLKETFYTIYKFEFDKTKMMSSRVDHILHGRELLKAEPHRDYKFRSDAVLPWGSLTKIKKCYSNQNQDAVLQHFQDQGSNEKRKYFARGHLAANADFVSQDEQKASYSFANVAPQWQAFNNGNWKKLENRIRTIAMKKEATLRVVTGTSTGLLKLNSSSGQLENVCLCDGEPPCVPLFFWKVTPALRQAFLMPNHPAAGISARIEELAHPCPNWAGFSGAEEKTRGALYCLTPGD LCGVEPKACGYKDLMGlass Shrimp Putative Nuclease:

Source: Eukaryota; Metazoa; Arthropoda; Crustacea; Malacostraca;Eumalacostraca; Eucarida; Decapoda; Pleocyemata; Caridea; Palaemonoidea;Palaemonidae; Palaemonidae sp.

Nucleic Acid Composition

TGTAGCGTGAAGACGACAGAAGTAATACGACTCACTATAGGGCAAGCAGTG (SEQ ID NO: 19)GTATCAACGCAGAGTACGCGGGCTCCTTGTTCAGAGCTTTAAAGTCATGGCTGGCAGGAGACAATTCTTCGTCTTATCTTTATATTTCGTGGCCTTTTGGAACCTTTCCAAAGGTCAAGATTGCGCCTGGGATAAGGATGCAGACTTCCCACTTACTCCTCCCCTTCTCTTGGATTCTTCCCTCAAGATGATATATCCAGTGTTAGAGGGATCCCTCAGGATGGTACGAGTAGCAGCTGGCAGCACTATCACCGTCGCATGTTCAGGGACGACAATTAGCTGCTCTAGGTCTCGAGGCTGTTGGAGGGAACCTGTGCTGGGGGCCCAACTGATCACTGGTGATGGCACTGATAATGCCCTAAATGAGTTGGGATGTGTGACGCCGCATCTGAGAGCTTGCAGAAGAACCTGGGTGCCTGTGAGATGCAGATCTTGGTACCTCCACGCAGTAGGATTTAATATTGCCACTACAGGTTCATTCCATGAATTCGAAAGTATATGTTTCGATCACGCTGCAGAGACTACTCTATACACAAAGCATACTCTCCATGGAGCCAACATCATAGCCAAAGACGTGGATCCTAGCAGACCACCCTTCAAGCCCGATACTGGATTCTTCACGGTCGAAGTCAATACCGTTTATACTCAGGGTTCACAGCTGGCCTTGATGGAACAGCTGCUGGTGATTCTGCACTGGCCAACCAGATCATTAATCCAGATCAAGAGCTGTTCATGTCTAGAGGTCACCTCTCTCCAGATGCTGACCATGTACTGATAGCTGAACAAGACGCAACTTATTACTTCATTAACGTTATGCCTCAGTGGCAGGCATTTAATAATGGAAACTGGAAGTACTTGGAATTTGCTGGCAGAGATCTTGCTGTAGCCCACGGCACTGACCTGACCGTCTACGATGGAGGTTGGGGAGTGCTGGAACTGGATGACATTAATGGAAATCCAGTACAGATTTACCTGGGACTCAGTGAAGGCAAAGAAGTTGTTCCAGCGCCTGCTCTCATGTATAAGATTCTGCACGAAGAAAGCACTAACCGAGCTGCAGCTGTTATAGGCATCAACAACCCCCACATTACAGTGGCTCCAACTCCTATTTGCACTGATATCTGCTCCAGTCTTACATGGATTGACTTCGACATTACTGACCTCTTCCGTGGTTTTACATACTGCTGCACCGTTGATGATCTCAGAGCAGCCATTCCTCACGTTCCTGATCTTGGAAATGTTGGTCTTTTGGACAGTTAAACATCCGTGACACTCTGTGAAAGAGGATCAGTTGTCGTGGGAATTGTTATAAATGAATAAATAATGACTACAGTAAAAAAAGAAAAA AAAAAAAAAAAAAAAAmino Acid Composition

MAGRRQFFVLSLYFVAFWNLSKGQDCAWDKDADFPLTPPLLLDSSLKMIYPVLE (SEQ ID NO: 20)GSLRMVRVAAGSTITVACSGTTISCSRSRGCWREPVLAAQLITGDGTDNALNELGCVTPHLRACRRTWVPVRCRSWYLHAVGFNIATTGSFHEFESICFDHAAETTLYTKHTLHGANIIAKDVDPSRPPFKPDTGFFTVEVNTVYTQASQLALMEQLLGDSALANQIINPDQELFMSRGHLSPDADHVLIAEQDATYYFINVMPQWQAFNNGNWKYLEFAGRDLAVAHGTDLTVYDGGWGVLELDDINGNPVQIYLGLSEGKEVVPAPALMYKILHEESTNRAAAVIGINNPHITVAPTPICTDICSSLTWIDFDITDLFRGFTYCCTVDDLRAAIPHVPDLGNVGLLDSMangrove Fiddler Crab Putative Nuclease:

Source: Eukaryota; Metazoa; Arthropoda; Crustacea; Malacostraca;Eumalacostraca; Eucarida; Decapoda; Pleocyemata; Brachyura; Eubrachyura;Ocypodoidea; Ocypodidae; Ocypodinae; Uca complex; Uca; Uca crassipes

Nucleic Acid Composition

AAGCAGTGGTATCAACGCAGAGTACGCGGGGGGGAGAAGCACTGCGCTGA (SEQ ID NO: 21)GAGAAGCAGAGAGGAAATGGATCTCCGACGAAGATTCTCCCGGACCTTACAACTGGTAGTCCTTCTCTTCGCCTGTGCAAGCAATTGCTTTGGATGCGAGTGGGACAAAGACTTGGACTTCCCTGAACACCCGCCGCTCATCATTAACAACCAGCTAGATTTCGTGCTGCCGGTGTTGGAGGGAGTCAACAGGGTGGTGAGGGTGGCAGAGGGAGAAACCGTGACTCTGGCGTGCTCTGGTAGCGAGTTGGTAAATCTTGGGGAAGCAGAGGTGCAGGCTCGGTGCCTCAGCAGTGGCCTCCTAACGATCGGTGATGCAGAGTGGGACTTGGcGAGCCTtGGTTGCagCAGTGATGTAAAAGAGACCATTTTCCGCGACCTGGGGACCTGCGGCGCCGGTGGTGTCGGGATCCTAAATGGCATTGGCTTCCAGATTTTCAGTCTCAACTACGACAAAGTGATCATTAACGTTTGCTTCGAAGCAGCTTCCGAGACGACCCTCTTCACTGATCACATCCTCCACGGCGCCGACATCGCCGCTAAGGACGTAGAGGCGTCCAGGCCGTCCTTTAAGACTTCCACAGGGTTCTTCAGTGTCTCTATGAACACCGTGTATTCGCAGAACTCGCAACTCCAACTCATGACTAGTATTCTCGGAGACGAGGACCCCGCCAATACAATTATTGACCCTTCCAAACAACTATACTTCGCAAAGGGTCACATGTCTCCTGACGCCGGTTTCGTGACTATAGCAAGCCAGGATGCCACCTATTACTTCATCAATGCCTTGCCACAGTGGCAGGCCTTCAACAATGGCAACTGGAAGTATCTGGAGACTAACACGCGAAATCTGGCAATGAAGAAGGGACGCGACCTTCGGGTGTACAGTGGTGGGTGGGATGTCCTGGAGCTGGACGACATTAATGGCAATCCCGTGAAGGTCTTCCTGGGACTGACAGAAGGCAAGGAGGTAGTGCCCGCGCCTGCCATCACCTGGAAGGTGGTACACGATGAGTCCACTAACTGCGCCGTGGCCGTAGTGGGCGTCAACAACCCGCATCTCACCGCCGCCCCCGCCACGCTTTGTGAAGACCTGTGTTCCTCGCTCTCCTGGATCACCTTCGACGTTAGCAGTCTCGCAAGCGGGTACACCTACCGCTGTTCCGTGGCGGAACTGCGCGCCTCGGTGCCCCACGTTCCTGACTTAGGCAATGTCTGTCTTCTCACCGACTAAAGACGAAACACATGTTGGAGTGACCCAGTAAATGGACAGTGGTGACATCAGGTTGACCTAATCATAATAGTGTCATCATGCTAAAAAAAAAAAAmino Acid Composition

MDLRRRFSRTLQLVVLLFACASNCFGCEWDKDLDFPEHPPLIINNQLDFVLPVLE (SEQ ID NO: 22)GVNRVVRVAEGETVTLACSGSELVNLGEAEVQARCLSSGLLTIGDAEWDLASLGCSSDVKETIFRDLGTCGAGGVGILNGIGFQIFSLNYDKVIINVCFEAASETTLFTDHILHGADIAAKDVEASRPSFKTSTGFFSVSMNTVYSQNSQLQLMTSILGDEDPANTIIDPSKQLYFAKGHMSPDAGFVTIASQDATYYFINALPQWQAFNNGNWKYLETNTRNLAMKKGRDLRVYSGGWDVLELDDINGNPVKVFLGLTEGKEVVPAPAITWKVVHDESTNCAVAVVGVNNPHLTAAPATLCEDLCSSLSWITFDVSSLASGYTYRCSVAELRASVPHVPDLGNVCLLTDII. DSN Preference (DSNP) Assay—Methods of Detection of the Sequence(s)and Sequence Variants in Nucleic Acid SamplesA. General Description

Under certain conditions, the subject nucleases specifically recognizeand cut perfect short DNA/DNA (and/or DNA/RNA) duplexes with higheractivity then non-perfect duplexes of the same length. Thus, subjectnucleases are capable of discriminating between a single nucleotidemismatch in the short duplex (which is not cut by the enzyme) and aperfect duplex (which is cut by the enzyme) (FIG. 1). This ability ofsubject nucleases in the employed in the methods for detection ofnucleic acid sequence(s) and sequence changes including single base isdescribed below.

A schematic diagram of the DSNP assay is provided in FIG. 8. In thesemethods, the sample of nucleic acids to be tested is mixed with subjectnuclease and labeled probe oligonucleotide. Probe oligonucleotide canform perfectly matched duplexes with sequence (or sequence variant) ofinterest. In certain embodiments, probe oligonucleotide is labeled byfluorescent donor and fluorescent quencher (or acceptor) pair and cangenerate specific signal after cleaving. In other embodiments, probeoligonucleotide is labeled by another label known in the art asdescribed previously.

The resultant mixture is incubated under conditions sufficient forgeneration of the duplexes between target sample nucleic acids and probeoligonucleotide. The perfectly matched duplexes generated are cleaved bythe subject nuclease. Examples of the appropriate conditions aredescribed in details in Examples section below.

After incubation, the cleaving of the probe oligonucleotide is examined.The manner in which the cleaved oligonucleotide probes are detectednecessarily depends on the nature of the oligonucleotide probe label.For example, where the detectable label is an isotopic label positionedat one end of the probe, one can assay for detectably labeled fragmentsthat are shorter than the full-length probe size and, in this manner,detect the presence of cleaved probes. In certain embodiments, thechange of the fluorescence intensity of the testing sample is measuredand compared with the change of the fluorescence intensity of thepositive and negative control samples that are known to comprise or notcomprise sequence of interest. The fluorescence intensity change in thetesting sample that exceed the fluorescence intensity change in thenegative control and comparable with fluorescence intensity change inthe positive control sample support is an indication of the presence ofthe sequence of interest in the testing sample.

As mentioned above, the above general methods can be used to detect thepresence of a single nucleic acid sequence variant of interest in asample or a plurality of different nucleic acid sequences in a sample(e.g. for testing both allelic variants at one or for testing severalmicrobial strains, etc.). When a plurality of different nucleic acidsequence variants are to be detected in one tube, a specific probeoligonucleotide for each sequence (sequence variant) to be detected isused. In this case, the only limitation is that a labeling system, i.e.,signal producing system of one or more entities, should be employed thatprovides for ready differentiation of different oligonucleotide probesto different target nucleic acid sequence variants (FIG. 9).Alternatively, in the case of the array of the subject invention(described below), the oligonucleotides may contain a same label,because the signals for different sequence variants would be differentby their positions.

For these DSNP assay methods, the following starting materials may beused:

1-Nucleic Acid Sample Containing Sequence(s) of Interest.

The nucleic acid sample being analyzed can be obtained from any sourceand can be obtained from these sources using standard methods. Thesample might include single-stranded DNA or double-stranded DNA (e.g.genomic DNA) in linear or circular form. Depending on the particularinterest, the sample may be treated by different methods known in theart, e.g. mechanical shearing, restriction enzyme digest, etc. In someembodiments, the sample may be from a synthetic source. In someembodiments, nucleic acids in the sample may be amplified by PCR orother methods known in the art. In some embodiments, nucleic acids to betested are RNA, natural or synthesized (for example synthesized from T7promoter-containing PCR amplified DNA by known methods). In someembodiments, in this case the prior step is preparation of the firststrand cDNA that may be performed using any method known in the art. Inother embodiments. In other embodiments, nucleic acid sample is RNA thatis used directly from its naturally occurring source or synthesized. Incertain embodiments, region comprising sequence(s) of interest isamplified by PCR using specific PCR primers. The length of the PCRproduct may vary, but usually range from 10 to 1000 bp, more usuallyfrom 60 to 500 bp.

2-Subject Nuclease.

A variety of different nucleases may exhibit the specific propertiesdescribed above under specific cleavage conditions and thus may beemployed in the subject methods. Representative nucleases of interestinclude but are not limited: cation-dependent endonucleases fromdifferent sources including DNAase K from Kamchatka crab (Menzorova, etal., Biochemistry (Moscow), vol. 58 (1993) (in Russian) pp. 681 to 691;Menzorova, et al., Biochemistry (Moscow), vol. 59 (1994) pp 321 to 325),Dnase I family members (like well known bovine DNAase I)(Liao T H. MolCell Biochem 1981 Jan. 20; 34(1):15-22), non-specific nucleases likeshrimp nuclease (Chou & Liao; Biochemica et Biophysica Acta, vol. 1036(1990) pp 95 to 100; Lin et al., Biochemica et Biophysica Acta, vol.1209 (1994) pp 209 to 214; Wang et al., Biochem. J., vol 346 (2000) pp799 to 804), and Ca²⁺,Mg²⁺-dependent endonuclease from sea-urchin(Menzorova, N. I., Rasskazov, V. A. Biokhimiia (Rus) 1981; vol 46 pp 872to 880), and the like. Of particular interest in many embodiments arethe novel specific nucleases described herein, including the crabduplex-specific nuclease (crab DSN).

-   3—Buffer for the Subject Nuclease (DSN Buffer).-   4—Probe Oligonucleotides.

The specific complementary short oligonucleotide is designed for eachsequence or sequence variant to be tested. The length of the probeoligonucleotide typically range from 9 to 30 nt, usually 10-15 nt ifnucleic acid samples are DNA and 15-20 nt if nucleic acid samples areRNA. Each probe oligonucleotide is labeled by detectable label as knownin the art. Preferably labeling is performed using fluorescence donorand quenching agent (or fluorescence acceptor). Preferably theoligonucleotide probes are labeled using fluorescence donors withdifferent color to generate sequence-specific fluorescence aftercleaving. In other words, each probe oligonucleotide is labeled togenerate fluorescence at specific wavelengths after cleaving. Theprimers and oligonucleotides used in the methods of the presentinvention are DNA, and can be synthesized using standard techniques.

B. Examples of DSNP Assay.

-   1. Detection of Point Mutations (SNPs) on DNA Samples (e.g. cDNA or    Genomic DNA) in Solution with Fluorescence-Labeled Oligonucleotide    Probes.-   1.1. Initially, the DNA fragments containing the SNP site of    interest are amplified by PCR. Following amplification, an aliquot    of the PCR reaction is mixed with DSN and with two probe    oligonucleotides labeled with the fluorescence donor (at the 5′ end)    and quencher (at the 3′ end). Each signal probe generates    fluorescence at specific wavelengths after cleaving. The first    oligonucleotide is complementary to the wild-type sequence, while    the second is complementary to the mutant sequence. The mixture is    incubated with DSN during which the nuclease cleaves the PCR product    to generate short DNA fragments that can effectively hybridize with    signal probes. All perfectly matched duplexes generated by the DNA    template and signal probes are cleaved by DSN to generate    sequence-specific fluorescence. Incubation conditions are dependent    at least in part on probe oligonucleotide length and compositions.    In certain embodiments (when 10 nt probe oligonucleotides is used),    the incubation is performed at temperature range from 29 to 37° C.    (e.g. at 35° C.) for a period 2-6 h (or longer).-   1.2. To improve signal intensity, the DSN cleavage reaction in the    method described in section 1.1 is performed in the presence of    exonuclease-deficient Klenow fragment (KF(exo-)), which catalyzes    strand displacement DNA synthesis by extension of the 3′-ends    generated in the PCR fragment upon DSN nicking activity. Displaced    DNA strands are involved in a genotyping reaction that results in a    5-20 times increase in the specific fluorescent signal. In certain,    the incubation is performed at temperature range from 29 to 37° C.    (e.g. at 35° C.) for a period 30 min-2 h.-   1.3. Initially, the DNA fragment containing the SNP site of interest    is amplified by PCR. Following amplification, an aliquot of the PCR    reaction is mixed with DSN and with two fluorescence-labeled    oligonucleotide probes as in section 1.1. The resulted mixture is    incubated at temperature of fragmentation that is optimal for DSN    cleaving (usually from 50 to 65° C.) for period sufficient for    cleaving of the PCR product to generate short DNA fragments (usually    from 7 to 20 nt). The incubation time may vary, but usually ranges    from 10 min to 1 h, more usually 20-30 min. After, the resultant    mixture is incubated at annealing temperature (usually 35° C. for    10-mer oligonucleotide probes) for a period sufficient for    hybridization of probe oligonucleotides and target nucleic acid    fragments and for cleaving of all perfectly matched duplexes    generated. The incubation time may vary, but usually ranges from 5    min to 2 hrs, more usually from 10 to 30 min.    This method can be employed to either qualitatively or    quantitatively detect the ratio of the mutant and wild-type sequence    variants in the sample. The ratio of two labels reflects the ratio    of mutant nucleic acids and wild type nucleic acids in the sample.-   1.4. The PCR products (obtained as described in previous examples)    are first incubated with DSN at temperature of fragmentation without    probe oligonucleotides. Probe oligonucleotides are added to the    reaction during or after this step and the resultant mixture is    incubated at annealing tempereature. This method can be employed to    either qualitatively or quantitatively detect the ratio of the    mutant and wild-type sequence variants in the sample. The ratio of    two labels reflects the ratio of mutant nucleic acids and wild type    nucleic acids in the sample.-   1.5. In the methods described above (sections 1.1 to 1.4), two    fluorescent dyes are employed to distinguish between wild-type and    mutant sequences in one tube. However, the assays may be also    performed when both probe oligonucleotides generate one type of    fluorescence. In this case, wild-type and mutant sequence-specific    probe oligonucleotides are mixed with DNA substrates in two separate    tubes.-   1.6. In some cases (e.g. DNA samples with low complexity), the    initial PCR amplification step may be excluded from the methods    described above (sections 1.1 to 1.5). For example, plasmid DNA    comprising sequence of interest may be tested in DSNP assay directly    without PCR amplification. Purified PCR products may also be used in    the assay.    The SNP detection using DSNP assay was examined on several models,    using homozygous and heterozygous DNA samples. All the results    obtained with the DSNP assay were confirmed by DNA sequencing.

The DSNP assay variant described in the section 1.1 was performed on twoPCR fragments of 144 bp that are different in one nucleotide positionwere prepared: Firstsequence—FT7normD—agtacgctcaagacgacagaagtacgcccgggcgtactctgcgttgttaccactgctttggagctccaattcgccctatagtgagtcgtattattctgtcgtcttcacgctaca (SEQ ID NO:23) and Secondsequence—TT79cD—agtacgctcaagacgacagaagtacgcccgggcgtactctgcgttgttaccactgctttggagctccaattcgccctgtagtgagtcgtattattctgtcgtcttcacgctaca (SEQ ID NO:24). Each fragmentwas amplified to concentration of about 10 ng/mkl and then mixed withpair of fluorescently labeled oligonucleotides: TT79cD sequence specific5′-TAMRA-GCCCTGTAGT-DABCYL-3′ (SEQ ID NO:25) and FT7normD sequencespecific 5′-Fluorescein-GCCCTATAGT-DABCYL-3′ (SEQ ID NO:26). Reactionmixture (20 mkl) containing 15 mkl PCR reaction (in Advantage KlenTaqPolymerase buffer, Clontech), 5 mM MgCl₂, 0.25 mkM each labeledoligonucleotide, 0.4 Kunitz-units crab DSN was incubated at 30° C. for 3h. Analysis of resulted fluorescence showed strong FT7normD specificsignal in FT7normD sequence-containing sample and TT79cD-specific signalin TT79cD sequence-containing sample (FIG. 10).

To demonstrate the DSNP assay on PCR products with different size, PCRproducts of 952, 534 and 69 bp comprising C- or T-variants of the 7028C-T mitochondrial polymorphous site (in COX1 gene) were generated andused with a T-variant specific probe oligonucleotide in the DSNP assayas described in the section 1.2. PCR reactions were performed with theAdvantage 2 PCR Kit (Clontech). Each PCR reaction (25 μl) contained 1×Advantage 2 Polymerize mix (Clontech), 1× reaction buffer, 200 μM dNTPs,0.3 μM each gene-specific primer and 10 ng human total DNA. Fragments(69, 534, and 952 bp) comprising the 7028 C-T site of the COX1 gene wereamplified using three pairs of primers at the following positions: Dir1(6540-6559) and Rev1 (7492-7473); Dir2 (6792-6811) and Rev2 (7345-7326);Dir3 (6991-7010) and Rev3 (7060-7041) (primer positions are specifiedaccording to GenBank Accession Number NC_(—)001807). In each case, 26PCR cycles were performed at 95° C. for 7 s, 63° C. for 20 s and 72° C.for 30 s.

For genotyping reaction, in each case, a 5 μl aliquot of PCR productscontaining about 75 ng DNA was mixed with 1.5 μl 10×KF(exo-) buffer(Fermentas MBI), probe oligonucleotide 5′-Fl-gtgAgctaca-DABCYL-3′ (SEQID NO:27) (to a final concentration of 0.3 μM), 0.5 Kunitz unit crabDSN, 2 U KF(exo-) (Fermentas MBI) and milliQ water (to a final volume of15 μl), and incubated for 1 h at 35° C. Clear results were obtained withall PCR products as shown at photographs (FIG. 11). Photographs wereobtained using an Olympus SZX12 fluorescent stereomicroscope (FIG. 11A)and using a Multi Image Light Cabinet (Alpha Innotech Corporation) underUV light (FIG. 11B).

The DSNP assay scheme without PCR stage (as described in the section1.6) was demonstrated with plasmid DNA (about 4 kb) comprising a 69 bpCOX1 fragment (FIG. 11A). For genotyping reaction, 5 mkl containingabout 500 ng plasmid DNA in 1×DSN buffer was mixed with 1.5 μl10×KF(exo-) buffer (Fermentas MBI), probe oligonucleotide5′-Fl-gtgAgctaca-DABCYL-3′ (SEQ ID NO:28) (to a final concentration of0.3 μM), 0.5 Kunitz unit crab DSN, 2 U KF(exo-) (Fermentas MBI) andmilliQ water (to a final volume of 15 μl), and incubated for 1 h at 35°C. Photographs were obtained using an Olympus SZX12 fluorescentstereomicroscope.

We further employed the DSNP assay (described in the section 1.2) withtwo fluorescent dyes to detect specific polymorphisms in genetic locicontributing to susceptibility to some diseases. We analyzed prothrombin20210 G-to-A polymorphism associated with an increased risk of venousthrombosis and myocardial infarction (Poort, S. R., Rosendaal, F. R.,Reitsma, P. H., Bertina, R. M. Blood 1996, 88: 3698-3703.), C677Tpolymorphism of the MTHFR gene associated with increased levels of totalplasma homocysteine, a risk factor for coronary artery disease (Frosst,P., Blom, H. J., Milos, R., Goyette, P., Sheppard, C. A., Matthews, R.G., Boers, G. J., den Heijer, M., Kluijtmans, L. A., van den Heuvel, L.P., et al. Nat. Genet. 1995, 10: 111-113), and p53 C309T polymorphismassociated with carcinomas (Abarzua, P., LoSardo, J. E., Gubler, M. L.,Neri, A. Cancer Res. 1995, 55: 3490-3494) in homozygous and heterozygousDNA samples (FIG. 12). DSNP assay was performed as described inexample 1. The following PCR conditions and gene-specific primers wereemployed: p53 (C309T polymorphism): 30 PCR cycles (95° C. for 7 s; 65°C. for 20 s; 72° C. for 20 s) were performed with primers5′-aaggggagcctcaccacg-3′ (SEQ ID NO:29) and 5′-ccacggatctgaagggtgaa-3′(SEQ ID NO:30); prothrombin (20210 G-to-A polymorphism): 30 cycles (95°C. for 7 s; 63° C. for 20 s; 72° C. for 20 s), primers5′-atggttcccaataaaagtgac-3′(SEQ ID NO:31) and 5′-aatagcactgggagcattga-3(SEQ ID NO:32); MTHFR(C677T polymorphism): 30 PCR cycles (95° C. for 7s; 63° C. for 20 s; 72° C. for 20 s), primers5′-cttgaaggagaaggtgtctg-3′(SEQ ID NO:33) and 5′-aagaaaagctgcgtgatgatg-3′(SEQ ID NO:34). The following signal probes were used: p53 (C309Tpolymorphism): p53WT (wild-type-specific)—5′-Fl-tggGcagtgc-DABCYL-3′(SEQID NO:35) and p53M (mutant-specific)—5′-TAMRA-tggAcagtgc-DABCYL-3′ (SEQID NO:36); prothrombin (20210 G-to-A polymorphism):F2WT—5′-Fl-gctCgctgag-DABCYL-3′ (SEQ ID NO:37) andF2M—5′-TAMRA-gctTgctgag-DABCYL-3′(SEQ ID NO:38); MTHFR(C677Tpolymorphism): MTHFRWT—5′-Fl-tcgGctcccg-DABCYL-3′ (SEQ ID NO:39) andMTHFRM—5′-TAMRA-tcgActcccg-DABCYL-3′(SEQ ID NO:40).

DSNP assay described in the section 1.3 was used for testing of theFactor V Leiden polymorphism G1691A in homozygous and heterozygous DNAsamples (FIG. 13). Leiden mutation in factor V gene (Bertina et al.,Nature 1994; 369: 64-7) is the most common genetic abnormalityassociated with hereditary thrombophilia. DNA samples were used for PCRwith factor V specific primers (5′-tactaatctgtaagagcagatc-3′ (SEQ IDNO:41) and 5′-gttacttcaaggacaaaatacc-3′ (SEQ ID NO:42)) as described inexample 1. A 5 μl aliquot of PCR products containing about 75 ng DNA wasmixed with 1.5 μl 10×DSNP buffer (1× buffer contains 50 mM Tris-HCl, 8.0and 5 mM MgCl₂), probe oligonucleotides (to a final concentration of 0.3μM), 1 Kunitz unit DSN and milliQ water (to a final volume of 15 μl),and incubated for 15 min at 60° C. and 10 min at 35° C. Fluorescenceintensity was measured on a spectrofluorimeter Cary Eclypse (Varian).Plates were photographed using an Olympus SZX12 fluorescentstereomicroscope. The following probe oligonucleotides were used: FV-WT5′-Fl-aggcgaggaa-DABCYL-3′ (SEQ ID NO:43) and FV-M5′-TAMRA-aggcaaggaa-DABCYL-3′ (SEQ ID NO:44).

Quantitative testing using DSNP assay as described in the section 1.3was also demonstrated (FIG. 14).

DNA samples containing normal and mutant sequence of the ApoE gene(position 388) were used for PCR with ApoE specific primers(5′-gcggacatggaggacgt-3′ (SEQ ID NO:45) and 5′-ggcctgcacctcgccgcggta-3′(SEQ ID NO:46)). Resulted PCR amplified fragments were mixed indifferent proportions as follow:

Sample Normal Mutant number sequence, % sequence, % 1 100 0 2 80 20 3 6040 4 40 60 5 20 80 6 0 100 7 0 0 (negative control)A 5 μl aliquot of PCR products containing about 50 ng DNA was mixed with2 μl 10×DSNP buffer, probe oligonucleotides (to a final concentration of0.25 μM), 1 Kunitz unit DSN and milliQ water (to a final volume of 20μl), and incubated for 15 min at 60° C. and 10 min at 35° C. Thefollowing probe oligonucleotides were used: ApoEWT5′-Fl-cgtgcgcggc-DABCYL-3′ (SEQ ID NO:47) and ApoEM5′-TAMRA-cgtgtgcggc-DABCYL-3′ (SEQ ID NO:48). Fluorescence intensity wasmeasured on a spectrofluorimeter Cary Eclypse (Varian).2. Detection of Nucleic Acid Analytes in DNA Samples (e.g. cDNA orGenomic DNA) in Solution with Fluorescence-Labeled OligonucleotideProbes.DSNP assay is applicable for the nucleic acid analyte detection (forexpression profiling, detection of the bacterial or viral species andstrains in complex DNA samples, e.g. for diagnostic purpose, detectionof a specific PCR products in the PCR mixture without electrophoresis,etc.) For analysis of each nucleic acid analyte a short oligonucleotide,that is capable to hybridize nucleic acid analyte, is used. Theoligonucleotide is labeled at each end by a fluorescence donor andquenching agent pair. The DSNP assay for nucleic acid analyte detectionis carried out using any protocol described in the previous section(section 1 of the Examples). In certain embodiments, the prioramplification of the fraction enriched in the sequence(s) of interest isperformed, e.g. using PCR with gene-specific primers. The detection of asignal level that exceeds the signal level in sample incubated withoutnuclease is an indication of the presence of the nucleic acid analyte insample.

To quantitative assessment of the nucleic acid sequence in the sample,there are two variants: (1) introduction in the nucleic acid sample theendogenous nucleic acid (control nucleic acid) in known concentration;(2) using the nucleic acid of known concentration that is present in asample as control nucleic acid. For quantitative analysis theoligonucleotide specific to the control nucleic acid, which ispreferably labeled by another label then label for nucleic acid analyte,is added to reaction mixture. Comparison in cleavage rate of theseoligonucleotides allow to define quantity of nucleic acid analyte in thesample. If both oligonucleotides are labeled by the same label, analysismay be performed at separate reactions.

3. DSNP Assay On RNA Samples In Solution With Fluorescence-LabeledOligonucleotide Probes.

The DSNP assay is also applicable for detection of sequences or sequencevariants directly in RNA nucleic acids. As example, RNA to be analyzedis a synthetic RNA prepared using transcription from T7 promoter. Inthis method, 50-150 nt RNA containing the sequence(s) of interest ismixed with fluorescence labeled sequence-specific probeoligonucleotide(s) of 15-25 nt long. Cleavage reaction is performed in15-20 mkl containing 50 mM Tris-HCl (pH range from 7,5 to 8), at least100 ng RNA, 5-7 mM Mg²⁺, 0.3 mkM of each labeled probe oligonucleotide,0.4-1 Kunitz-units of DSN. SNP detection is performed as described inthe section 1.1 of the Examples.The method was tested in a model experiment (FIG. 15). Fragment of p53cDNA was amplified by PCR with gene-specific primers:309dir—5′-AGGGGAGCCTCACCACG-3′ (SEQ ID NO:49) and309rev—5′-CCACGGATCTGMGGGTGAA-3′ (SEQ ID NO:50) and then is ligated withT7 promoter containing oligonucleotide. Following PCR reactions wereperformed to prepare two PCR fragments the first from 309dir and T7primers and the second—from 309rev and T7 primers. These fragments wereused for RNA synthesis from T7 promoter. One of the resulted syntheticRNA corresponds to sence and another—to antisence RNA of p53 gene. TheseRNAs were mixed with fluorescently labeled (TAMRA at 5′end and quencherDABCYL at 3′end) oligonucleotide (15 nt) that is complemented to senseRNA and incubated with crab DSN. Reaction was performed in 20 mklcontaining 50 ng synthetic RNA; 25 mM Tris-HCl, pH7.8, 5 mM MgCl₂, 0.25mM fluorescent oligonucleotide. Reaction was carry out for 3 h at 35° C.and then was stopped by EDTA. Fluorescence intensity change in thereaction containing sense RNA was significant higher than fluorescenceintensity change in the reaction with antisense RNA (FIG. 16).4. DSNP Assay for Analysis of Allele-Specific PCR Results.Allele-specific amplification (ASA), also known as amplification ofspecific alleles (PASA) and amplification refractory mutation system(ARMS), is a generally applicable technique for the detection of knownpoint mutations, small deletions and insertions, polymorphisms and othersequence variations. Several methods based on the ASA principle havebeen described in the art (Sommer, S. S., J. D. Cassady, J. L. Sobell,and C. D. K. Bottema. Mayo Clin. Proc. 1989, 64: 1361-1372; Newton C R,Graham A, Heptinstall L E, Powell S J, et al., Nucleic Acids Research,1989, Vol 17, Issue 7, pages 2503-2516; Bottema C D, Sommer S S. MutatRes 1993, 288 (1):93-102; Hodgson D R, Foy C A, Partridge M,Pateromichelakis S, Gibson N J. Mol Med 2002, 8(5):227-237; Dutton C,Sommer S S. Biotechniques 1991, 11(6):700-2; Rust S, Funke H, Assmann G.Nucleic Acids Res 1993, 21(16):3623-3629; Ye S, Dhillon S, Ke X, CollinsA R, Day I N. Nucleic Acids Res. 2001, 29(17):E88-8; Liu Q, Thorland EC, Heit J A, Sommer S S. Genome Res. 1997, 7(4):389-398; Myakishev M V,Khripin Y., Hu S, Hamer D. Genome Res. 2001, 11:163-169). DSNP assay isapplicable for monitoring of the formation of allele-specific PCRproducts during the ASA (FIG. 16). This method utilizes allele-specificPCR primers, each of that contains a universal 5′-tail short uniquesequence (e.g. 10-15 nt) that becomes part of the PCR product onamplification.

PCR products may be obtained using any ASA modification. Afteramplification, PCR products are mixed with DSN and two probeoligonucleotides labeled with the fluorescence donor and quencher asdescribed above. Each probe generates fluorescence at specificwavelengths after cleaving. The first probe coincides with the uniqueuniversal sequence of the one allele-specific primer and the secondcoincides with the unique sequence of the other allele-specific primer.The mixture is incubated with DSN, during which the nuclease cleaves thePCR product to generate short DNA fragments that can effectivelyhybridize with probe oligonucleotides. Probe oligonucleotides anneal tothe 5′-tail universal sequences in the PCR products. All perfectlymatched duplexes generated by the DNA template and probeoligonucleotides are cleaved by DSN to generate allele-specificfluorescence.

Cleavage reaction with DSN for analysis of ASA results may be performedusing any protocol described above in the section 1 of the Examples.Probe oligonucleotides of the present example may vary but typicallyrange from 9 to 25 nt (usually 10-15 nt).

The method was tested in a model experiment for the analysis of the 7028C-T SNP in the COX1 gene. The allele specific primers(MtL-C₅′-gccctgtagtacacgtactacgttgttgcc-3′ (SEQ ID NO:51) and MtL-T5′-gctcgctgagacacgtactacgttgttgct-3′) were used for PCR with commonprimer Mit70Rev 5′-acagctcctattgataggac-3′ (SEQ ID NO:52). PCR wasperformed using the Advantage 2 PCR Kit (Clontech) as described above.25 PCR cycles were performed at 95° C. for 7 s, 62° C. for 20 s and 72°C. for 15 s. An 10 mkl of the PCR products containing about 75 ng DNAwas mixed with 1.5 μl 10×DSNP buffer, universal probe oligonucleotidesFL-gccctgtagt-Dab (SEQ ID NO:53) (C-variant specific) andTam-gctcgctgag-Dab (SEQ ID NO:54) (T-variant specific) to a finalconcentration of 0.25 μM, 1 Kunitz unit crab DSN, milliQ water (to afinal volume of 15 μl) and incubated at 60° C. for 10 min and then at35° C. for 10 min. Detection of fluorescence signals was performed underUV light. A clear result was obtained for both mutant and wild-typesequence comprising samples.

5. DSNP Assay on a Solid Phase.

The DSNP assay is suitable for a microarray format without the need forconsiderable modification. In this case, for each sequence (or sequencevariant) to be detected, a sequence-specific labeled probeoligonucleotide is designed. Probe oligonucleotides are immobilized onthe solid phase (microarray) through a spacer followed by a 10-25 ntspecific sequence, able to form perfectly matched duplexes withsequence(s) of interest (e.g. in the case of SNP detection, for each SNPtwo probes are used, which differ in the positions corresponding toallelic distinction). Each probe is labeled with a fluorescent donor anda quencher at the ends of the specific sequence, so that a fluorescencesignal is generated on a microarray after cleavage by DSN. All probeoligonucleotides may be labeled by one type of the label (e.g. one typeof fluorescent donor), because the signals for different sequencevariants would be different by their positions. A schematic diagram ofthe DSNP assay on a microarray is provided in FIG. 17.

6. DNA Sequencing

The DSNP assay is also useful for nucleic acid sequencing. To determinethe sequence of the nucleic acid of interest, the nucleic acid iscontacted under hybridizing conditions with a full set of every possibleoligonucleotide of the same length (for instance, 10 nucleotides long)placed on solid phase. Following treatment with an appropriate DNase, asdescribed in the section 1 of the Examples, above, cleavedoligonucleotides are detected as a means for determining whicholigonucleotides hybridized to the nucleic acid to be sequence. Thesequence of the test oligonucleotide is then compiled using knownprotocols of sequencing by hybridization, known to those of skill in theart and described in U.S. Pat. Nos. 5,525,464; 5,700,637 and 5,800,992,the disclosures of which are herein incorporated by reference.

III. Production of Normalized and Subtracted Libraries and Probes forDifferential Screening

A. General Description and Advantages Provided

The methods are based on selectively cleavage of DNA in DNA containingnucleic acid duplexes to retain the single stranded DNA of interest. Assuch, the subject methods can be used for the elimination of thefractions of redundant and/or common molecules of DNA duringnormalization and\or subtractive hybridization. The methods proposed aresimple and are applicable for full-length subtraction/normalization aswell as for subtraction/normalization on fragmented nucleic acids. Thesemethods are applicable for cDNA subtraction/normalization as well forgenome subtraction without any modifications. Also, during subtractivehybridization the enrichment in target molecules is accompanied by theequalization of target molecule concentration that prevents the raretranscript loss. Specific nucleases that may be used in the subjectmethods are duplex specific DNA nucleases. In certain embodiments,subject nucleases are heat stable nucleases like crab DSN.

B. Normalization

The following method based on the selective amplification of normalizedDNA ss-fraction formed by the partial reassociation of the denaturedds-cDNA may be employed to obtain equalized cDNA libraries. Thefollowing method does not include the physical separation of the ss- andds-fractions. ds-DNA fractions are degraded by DSN (double-strandedspecific DNAse) whereas ss fractions are not degraded. After DSNtreatment, the molecules of the ss-fractions are amplified by PCR.

The equalized cDNA library was obtained as outlined in the scheme (FIG.18). The scheme does not show the ds-cDNA synthesis that might beperformed by different ways. The method is applicable for bothfull-length and fragment (or digested) DNA. The normalization of genomicDNA samples might also be performed. The DNA must contain the knownterminal sequences (adaptors) that are ligated or are attached duringcDNA synthesis to DNA. Before normalization, DNA samples is purifiedusing methods known in the art, precipitated by ethanol and dissolved inhybridization buffer.

For the normalization, DNA is denatured and then is allowed to renature(hybridization step). Since the renaturation of an individual sequenceis the reaction of the second order in respect to the sequenceconcentration, the renaturation of abundant sequences are higher thanthose of rare sequences, which results in the equalization of the ss-DNAfraction.

After hybridization, the DNA sample is incubated with specific nucleaseto degrade ds-DNA fractions. Then inhibition of the nuclease activity(e.g. by heating) is then performed. After, residuary ss-DNA fractionmay be amplified by PCR with adaptor-specific primers. As a result,selectively amplified equalized cDNA is obtained.

The example of the appropriate protocol using crab DSN is follow: dscDNA (as noted above any other DNA samples may also be used) ispurified, precipitated by ethanol and dissolved in milliQ water to finalconcentration at least 25 ng/mkl. Of about 100-150 ng of purified DNA ismixed with hybridization buffer (50 mM HEPEC-HCl, pH8.3; 0,5 M NaCl),denatured (e.g. at 97° C. for 3 min) and allowed to renature (e.g. at70° C. for 5-6 h). After, 1 mkl 10×DSN buffer, 0.5-1 Kunitz-units DSNand milliQ water to final volume 10 mkl are added to the renatured DNAsample. Resulted mixture is incubated at 65° C. for 20-30 min. Afterincubation, DSN is inactivated by heating at 97° C. for 7-10 min.Reaction is diluted by milliQ water to 40 mkl, 1 mkl of the dilutedreaction is used for PCR to amplify normalized DNA.

This method was tested on cDNA sample prepared using Smart PCR cDNASynthesis Kit (CLONTECH) and amplified in PCR using Advantage™ 2 PCR Kitwith SMART II oligonucleotide primer. After 17 cycles of PCR, cDNA werepurified from unincorporated triphosphates and the excess of primersusing QJ Aquich PCR Purification Kit (Qiagen), precipitated by ethanoland diluted in water to final concentration 25 ng/mkl. 4 mkl DNAsolution was mixed with 1 mkl 4× hybridization buffer and denatured at98° C. for 3 min. Afterwards, cDNA was allowed for renature at 70° C.for 6 h and then mixed with 1 mkl 10×DSN buffer, 1 Kunitz-units crab DSNand milliQ water to final volume 10 mkl. Cleavage reaction was performedat 65° C. for 20 min. After incubation, DSN was inactivated by heatingat 97° C. for 7 min. Reaction was diluted by milliQ water to 40 mkl, 1mkl of the diluted reaction was used in PCR using Advantage™ 2 PCR Kitwith SMART II oligonucleotide primer (FIG. 19). To analyze theefficiency of normalization, the sequencing of the 400 independentclones from normalized and non-normalized libraries was performed. Apercent of the redundant clones in non-normalized library was 19%,whereas only 1% in normalized library.

C. Supernormalization

The method described in this section is a modification of the abovenormalization scheme. In this variant, DNA to be normalized (that may befirst strand cDNA, amplified cDNA, etc.) is mixed with driver nucleicacids (RNA or cDNA), derived from the same tissues. If the drivernucleic acids are DNA, the DNA preparation is performed so as DNA doesnot comprise adapter sequences. During hybridization, additionalhybrids, those are driver RNA-target DNA or driver DNA-target DNAhybrids, are generated that allow effective removal of abundantmolecules from ss-fraction. As was shown, DSN effectively cleave DNA inboth DNA-DNA and DNA-RNA duplexes. Thus, addition of driver nucleicacids leads to most efficient removal of abundant molecules (as comparedto example I) from ss-DNA fraction.

As an example, normalized cDNA from human liver RNA was prepared: polyARNA from human liver (CLONTECH) was used for full length first strandcDNA preparation by Smart PCR cDNA Synthesis Kit (CLONTECH). Reversetranscriptase was heat inactivated. First strand cDNA was purified andmixed with excess of liver polyA RNA, denatured and hybridized overnightat 69° C. in buffer for hybridization (CLONTECH). After hybridization,hybridizing DNA was diluted in DSN buffer and incubated with DSN at 65°C. for 30 min. The cleavage reaction was stopped by heating. Thenmixture was diluted to 40 mkl and 1 mkl was used in PCR with SMART IIoligonucleotide primer. 28 cycles of PCR were performed (FIG. 20, line2).

D. Subtractive Hybridization

A similar scheme was performed for subtractive hybridization (FIG. 21),only that driver nucleic acids is derived from other than tester DNAsource. The tester is genomic DNA or cDNA flanked by adapters. Driver isDNA (genome or cDNA) with no adapters or RNA (preferably mRNA).

-   1. At the first stage of the procedure the excess driver is added to    tester, the samples are melted and left to anneal. In the course of    hybridization, tester molecules that have their pair in driver for    the most part form hybrids driver-tester and thus become removed    from the ss-fraction. Meanwhile, target molecules are not affected    by the “driver pressing” and therefore ss-tester becomes enriched    with the molecules of this kind. Target molecules are also able to    form homohybrids with each other (reassociate). Reassociation    progresses much more rapidly for high abundant target molecules than    for low abundant ones. As a result, the concentrations of target    molecules in ss-tester become equalized. Driver abundant molecules    also form driver-driver hybrids and ss-driver molecules also remain.

After hybridization the sample is treated by DSN. DSN cleaves DNA inDNA-DNA and DNA-RNA hybrids. Thus, only ss-fractions remain. It wasshown that DSN has low activity to RNA both in ss-fraction and inRNA-RNA or RNA-DNA hybrids. Thus, in the scheme where driver is RNA,driver molecules are released from RNA-DNA hybrids after DNAdegradation. Moreover, DSN is thermostable and DSN treating is performedat the temperature where hybridization is effective. Thus, an additional“driver pressing” to tester occurs during DSN treating that leads tomost effective subtraction. After DSN treatment, target molecules(ss-tester fraction) might be amplified by PCR with adaptor-specificprimers. Driver molecules (if driver is DNA) are not amplified becausethey do not have these adapters.

-   2. In the course of hybridization, driver-tester hybrids are    generated most effectively by more abundant molecules. To increase    the power of subtraction additional freshly denatured driver is    added during hybridization. Excess of driver for redundant molecules    then appears much higher than at the beginning, because the majority    of them have been already removed from ss-fraction of tester. In    particular, the excess appears extremely high for molecules of    high-abundance class. This leads to the inversion of the original    disproportion in concentrations of redundant molecules: originally    high- and medium-abundant transcripts become almost entirely removed    from ss-tester.

As an example, the subtractive hybridization “liver-lung” was performed:full length first strand cDNA was prepared by Smart PCR cDNA SynthesisKit (CLONTECH) from human liver polyA RNA (CLONTECH). This first strandcDNA (tester) was mixed with 50× excess of lung polyA RNA, denatured at99° C. for 2 min and hybridized overnight at 69° C. in buffer forhybridization (CLONTECH). Hybridizing DNA was diluted in DSN buffer andincubated with DSN at 65° C. for 1 h. To stop the reaction, EDTA wasadded. Then mixture was diluted by ten folds and 1 μl was used in PCRwith SMART II oligonucleotide primer. 30 cycles of PCR were performed(FIG. 20, line 3).

It is evident that the above results and discussion that the subjectinvention provides an important new nuclease with novel activities thatfinds use in a variety of different applications. Therefore, the presentinvention represents a significant contribution to the art.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference. The citation of any publication is for its disclosure priorto the filing date and should not be construed as an admission that thepresent invention is not entitled to antedate such publication by virtueof prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1. An isolated divalent cation dependent thermostable nuclease with atleast 95% amino acid sequence identity to the amino acid sequence of anuclease encoded by the nucleic acid comprising SEQ ID NO:02.
 2. Thenuclease according to claim 1 wherein said nuclease hydrolyzes singlestranded nucleic acids and RNA in duplex nucleic acids with loweractivity than DNA in duplex nucleic acids.
 3. The nuclease according toclaim 1 wherein said nuclease cleaves deoxyribonucleic acid molecules inshort completely matched duplex nucleic acids with at least 10-foldhigher activity than in non-completely matched duplex nucleic acids ofthe same length.
 4. The nuclease according to claim 1 wherein saidnuclease is isolated from the Kamchatka crab.
 5. The nuclease accordingto claim 2 wherein said nuclease hydrolyzes single stranded nucleicacids and RNA in duplex nucleic acids with at least 10-fold loweractivity than DNA in duplex nucleic acids.
 6. The nuclease according toclaim 1 wherein said nuclease is recombinant.
 7. A method of selectivelycleaving deoxyribonucleic acid molecules in duplex nucleic acids in acomplex nucleic acid sample, said method comprising: contacting saidsample with a nuclease according to claim 1 under DSN conditions for aperiod of time sufficient for said duplex nucleic acids in said sampleto be selectively cleaved.
 8. The method according to claim 7, whereinsaid method comprises distinguishing duplex deoxyribonucleic acids fromsingle stranded deoxyribonucleic acids.
 9. The method according to claim7, wherein said method comprises distinguishing completely matchingduplex deoxyribonucleic acids that include at least one deoxyribonucleicacid molecule from non-completely matching duplex deoxyribonucleic acidsof the same length.
 10. A kit for use in performing a method accordingto claim 7, wherein said kit comprises: a nuclease according to claim 1;and instructions for practicing a method according to claim 7.